Substituted purine compounds

ABSTRACT

The present invention relates to substituted purine compounds. The present invention also relates to pharmaceutical compositions containing these compounds and methods of treating disorders in which DOT1-mediated protein methylation plays a part, such as cancer, by administering these compounds and pharmaceutical compositions to subjects in need thereof.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/902,150, filed on Dec. 30, 2015, now allowed and to be issued asU.S. Pat. No. 9,688,714 on Jun. 27, 2017, which is a U.S. National Phaseapplication, filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2014/045406, filed Jul. 3, 2014, which claims priority to, andthe benefit of, U.S. provisional application No. 61/842,701, filed Jul.3, 2013, the entire contents of each of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

Disease-associated chromatin-modifying enzymes (e.g., DOT1L) play a rolein diseases and disorders such as proliferative disorders, metabolicdisorders and blood disorders. Thus, there is a need for the developmentof small molecules that are capable of modulating the activity of DOT1L.

SUMMARY OF THE INVENTION

The invention provides substituted purine compounds, includingmetabolites of((2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol)(Compound A)

The compounds disclosed herein are useful for modulating the aberrantaction of epigenetic enzymes and are useful for treating a disease ordisorder in which an epigenetic enzyme plays a role. The presentinvention also provides pharmaceutically acceptable salts, and/or estersof these compounds.

In one aspect, the present invention features a compound of Formula (I)below.

In this formula, each of l, m, and n, independently is 0, 1, or 2, eachof p, q, r, t, u, v, and w, independently is 0 or 1, and the sum of l,m, n, p, q, r, t, u, v, and w is 1, 2, or 3.

One subset of the compounds of Formula (I) includes those of Formula(IA):

Another subset of the compounds of Formula (I) includes those of Formula(IB):

In another aspect, the present invention features a pharmaceuticallyacceptable salt or ester of a compound of Formula (I), (IA), or (IB)above.

In yet another aspect, the present invention features a compound ofFormula (II) below or a pharmaceutically acceptable salt or esterthereof.

In Formula (II), R₁ is unsubstituted t-butyl or t-butyl substituted withone or more substituents selected from hydroxyl and oxo (i.e., ═O), R₂is H, hydroxyl, unsubstituted i-propyl, or i-propyl substituted with oneor more hydroxyl, l′ is 0, 1, 2, or 3, each of m′ and n′, independentlyis 0, 1, or 2, each of p′, q′, r′, t′, u′, v′, and w′, independently is0 or 1, and when R₁ is t-butyl substituted with only one hydroxyl, R₂ isH, hydroxyl, or i-propyl substituted with one or more hydroxyl, and whenR₁ is unsubstituted t-butyl, then (i) R₂ is hydroxyl, or i-propylsubstituted with one or more hydroxyl, or (ii) the sum of l′, m′, n′,p′, q′, r′, t′, u′, v′, and w′ is 1 or greater.

One subset of the compounds of Formula (II) includes those of Formula(IIA):

Another subset of the compounds of Formula (I) includes those of Formula(IIB):

In yet another aspect, the present invention features a compound isselected from compounds 1-89, 101-104, and 107-114 and pharmaceuticallyacceptable salts thereof.

The invention also relates to a pharmaceutical composition of a compoundof any of the Formulae described herein and a pharmaceuticallyacceptable carrier.

The invention also relates to a pharmaceutical composition of a salt ofa compound of any of the Formulae described herein and apharmaceutically acceptable carrier.

The invention also relates to a pharmaceutical composition of a hydrateof a compound of any of the Formulae described herein and apharmaceutically acceptable carrier.

The present invention provides methods of treating or preventing adisease or disorder in which DOT1L plays a role such as cancer. Thepresent invention provides methods of treating cancer. The presentinvention also provides methods of preventing cancer. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of the compound of any of the Formulae describedherein. The cancer can be a hematological cancer. For example, thecancer is leukemia. Particularly, the cancer is acute myeloid leukemia,acute lymphocytic leukemia or mixed lineage leukemia.

The present invention provides methods of treating a disease or disordermediated by translocation of a gene on chromosome 11q23. The presentinvention also provides methods of preventing a disease or disordermediated by translocation of a gene on chromosome 11q23. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of the compound of any of the Formulae describedherein.

The present invention provides methods of treating a disease or disorderin which DOT1-mediated protein methylation plays a part or a disease ordisorder mediated by DOT1-mediated protein methylation. The presentinvention also provides methods of preventing a disease or disorder inwhich DOT1-mediated protein methylation plays a part or a disease ordisorder mediated by DOT1-mediated protein methylation. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of the compound of any of the Formulae describedherein.

The present invention provides methods of inhibiting DOT1L activity in acell. The method includes contacting the cell with an effective amountof one or more of the compound of any of the Formulae described herein.

Still another aspect of the invention relates to a method of reducingthe level of Histone H3 Lysine residue 79 (H3-K79) methylation in acell. The method includes contacting a cell with a compound of thepresent invention. Such method can be used to ameliorate any conditionwhich is caused by or potentiated by the activity of DOT1 through H3-K79methylation.

The present invention relates to use of the compounds disclosed hereinin preparation of a medicament for treating or preventing cancer. Theuse includes a compound of any of the Formulae described herein foradministration to a subject in need thereof in a therapeuticallyeffective amount. The cancer can be a hematological cancer. For example,the cancer is leukemia. Particularly, the cancer is acute myeloidleukemia, acute lymphocytic leukemia or mixed lineage leukemia.

The present invention provides use of the compounds disclosed herein inpreparation of a medicament for treating or preventing a disease ordisorder mediated by translocation of a gene on chromosome 11q23. Theuse includes a compound of any of the Formulae described herein foradministration to a subject in need thereof in a therapeuticallyeffective amount.

The present invention provides use of the compounds disclosed herein inpreparation of a medicament for treating or preventing a disease ordisorder in which DOT1-mediated protein methylation plays a part or adisease or disorder mediated by DOT1-mediated protein methylation. Theuse includes a compound of any of the Formulae described herein foradministration to a subject in need thereof in a therapeuticallyeffective amount.

The present invention provides use of the compounds disclosed herein forinhibiting DOT1L activity in a cell. The use includes contacting thecell with an effective amount of one or more of the compound of any ofthe Formulae described herein.

Still another aspect of the invention relates to a use of the compoundsdisclosed herein for reducing the level of Histone H3 Lysine residue 79(H3-K79) methylation in a cell. The use includes contacting a cell witha compound of the present invention. Such use can ameliorate anycondition which is caused by or potentiated by the activity of DOT1through H3-K79 methylation.

Also, the present invention provides a method for treating oralleviating a symptom of leukemia comprising administering to a subjectin need thereof a therapeutically effective amount of a compounddescribed herein. In one aspect, the leukemia is characterized by achromosomal rearrangement. In one aspect, the chromosomal rearrangementis chimeric fusion of mixed lineage leukemia gene (MLL) or partialtandem duplication of the MLL gene (MLL-PTD). In another aspect, thesubject has an increased level of HOXA9, Fms-like tyrosine kinase 3(FLT3), MEIS1, and/or DOT1L.

The present invention provides a method for treating or alleviating asymptom of leukemia comprising administering to a subject in needthereof a therapeutically effective amount of a compound describedherein, wherein the subject has an increased level of HOXA9, FLT3,MEIS1, and/or DOT1L.

The present invention provides a method for treating or alleviating asymptom of leukemia in a subject comprising: obtaining a sample from thesubject; detecting the level of HOXA9, FLT3, MEIS1, and/or DOT1L,wherein an increased level of HOXA9, FLT3, MEIS1, and/or DOT1L indicatesthe subject is responsive to a compound described herein; andadministering to the subject a therapeutically effective amount of saidcompound when said subject is responsive to said compound.

The present invention provides a method for treating or alleviating asymptom of leukemia in a subject comprising: obtaining a sample from thesubject; detecting the presence of a genetic lesion of MLL in thesample; and administering to the subject a therapeutically effectiveamount of a compound described herein when said genetic lesion ispresent in the sample. In one aspect, the genetic lesion is chimericfusion of MLL or MLL-PTD.

In any of the foregoing methods, the sample is selected from bonemarrow, peripheral blood cells, blood, plasma, serum, urine, saliva, acell, or a tumor tissue.

The present invention provides a method for treating a disorder mediatedby translocation, deletion and/or duplication of a gene on chromosome11q23, comprising administering to a subject in need thereof atherapeutically effective amount of a compound described herein.

In another aspect, the invention features a method of selecting atherapy for a subject having leukemia. The method includes the steps of:detecting the presence of a partial tandem duplication of the MLL gene(MLL-PTD) in a sample from the subject; and selecting, based on thepresence of the MLL-PTD, a therapy for treating leukemia. In oneembodiment, the therapy includes administering to the subject atherapeutically effective amount of a compound described herein. In oneembodiment, the method further includes administrating to the subject atherapeutically effective amount of a compound described herein. In oneembodiment, the leukemia is characterized by partial tandem duplicationof the MLL gene.

In another aspect, a method of treatment is provided for a subject inneed thereof, the method comprising the steps of: detecting presence ofa partial tandem duplication of the MLL gene (MLL-PTD) in a sample fromthe subject; and treating the subject based on the presence of MLL-PTDwith a therapy that includes administrating to the subject atherapeutically effective amount of a compound described herein. In oneembodiment, the subject in need thereof has leukemia that ischaracterized by partial tandem duplication of the MLL gene.

In another aspect, the invention features a method of selecting atherapy for a subject having leukemia. The method includes the steps of:detecting the level of HOXA9, FLT3, MEIS1, and/or DOT1L in a sample fromthe subject; and selecting, based on the presence of the increased levelof HOXA9, FLT3, MEIS1, and/or DOT1L a therapy for treating leukemia. Inone embodiment, the therapy includes administering to the subject atherapeutically effective amount of a compound described herein. In oneembodiment, the method further includes administrating to the subject atherapeutically effective amount of a compound described herein. In oneembodiment, the leukemia is characterized by partial tandem duplicationof the MLL gene. In another embodiment, the leukemia is characterized byoverexpression of HOXA9, FLT3, MEIS1 and/or DOT1L.

In yet another aspect, a method of treatment is provided for a subjectin need thereof, the method comprising the steps of: detecting the levelof HOXA9, FLT3, MEIS1, and/or DOT1L in a sample from the subject; andtreating the subject based on the presence of the increased level ofHOXA9, FLT3, MEIS1, and/or DOT1L with a therapy that includesadministrating to the subject a therapeutically effective amount of acompound described herein. In one embodiment, the subject in needthereof has leukemia that is characterized by partial tandem duplicationof the MLL gene. In another embodiment, the subject in need thereof hasleukemia that is characterized by overexpression of HOXA9, FLT3, MEIS1and/or DOT1L.

In addition, the invention provides methods of synthesizing theforegoing compounds. Following synthesis, a therapeutically effectiveamount of one or more of the compounds can be formulated with apharmaceutically acceptable carrier for administration to a mammal,particularly humans, for use in modulating an epigenetic enzyme. Incertain embodiments, the compounds of the present invention are usefulfor treating, preventing, or reducing the risk of cancer or for themanufacture of a medicament for treating, preventing, or reducing therisk of cancer. Accordingly, the compounds or the formulations can beadministered, for example, via oral, parenteral, otic, ophthalmic,nasal, or topical routes, to provide an effective amount of the compoundto the mammal.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are plots depicting the preclinical pharmacokinetics ofCompound A as determined in mouse, rat and dog, respectively. FIG. 1A isa concentration vs. time profile of plasma concentrations (mean±SD(n=3)) following IV bolus (5 mg/kg) administration to CD-1 mice (allformulated in 10% ethanol:90% saline). FIG. 1B is a concentration vs.time profile of plasma concentrations (mean±SD (n=3)) following IV bolus(1 mg/kg formulated in 0.4% HPBCD in saline) administration to SD rats.FIG. 1C is a concentration vs. time profile of plasma concentrations(mean±SD (n=3)) following IV bolus (1 mg/kg formulated in 10% ethanol:90% saline) administration to male Beagle dogs.

FIG. 2 is a plot of a concentration vs. time profile of plasmaconcentrations (mean±SD (n=3)) following IV infusion (4.7 mg/kg/day for7 days formulated in 10% PEG400:90% saline) administration to SD rats.

FIGS. 3A-3C are representative allometric plots for Compound A usingmouse, rat and dog PK data plotted with line of best fit: (A) unboundclearance (“CL”) vs. bodyweight (R²=0.9992); (B) product term of CL andmaximum life potential (“MLP”) vs. bodyweight (R²=0.9993); (C)steady-state volume of distribution (“VDss”) vs. bodyweight (R²=0.9999).

FIG. 4 is a time-invariant PK profile using the Wajima approach ofplasma concentration normalized for steady state concentration (“Css”)vs. time normalized for mean residence time (“MRT”) for each of thepreclinical species, mouse (solid squares), rat (open circles) and dog(solid triangles). Solid line is line of best fit of the aggregate data.

FIGS. 5A and 5B are representative HPLC-MS chromatograms for t=20 minand t=0 min incubations, respectively, of Compound A in mouse livermicrosomes supplemented with NADPH and UDPGA.

FIGS. 6A-6D are respectively LC-MS chromatogram, MS1 and MS2 spectra,and a scheme of proposed fragmentation pathways of Compound A.

FIGS. 7A and 7B are respectively MS2 spectra of Compound M1, M3, or M5(m/z 579) and Compound A (m/z 563) with proposed fragmentation pathways.

FIGS. 8A and 8B are respectively MS2 spectra of Compound M4 (m/z 521)and Compound A (m/z 563) with proposed fragmentation pathways.

FIGS. 9A and 9B are respectively MS2 spectra of Compound M6 (m/z 579)and Compound A (m/z 563) with proposed fragmentation pathways.

FIG. 10 is a scheme depicting the proposed major metabolic pathways ofCompound A in mouse, rat, dog and human liver microsomes supplementedwith NADPH and UDPGA.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides compounds that can be usedto selectively modulate the aberrant action of an epigenetic enzyme.Further, the compounds can be used to treat or prevent a disease statein a mammal caused or mediated by aberrant action of an epigeneticenzyme. The present invention includes pharmaceutically acceptablesalts, esters, and tautomers of these compounds.

The present invention provides novel substituted purine compounds,synthetic methods for making the compounds, pharmaceutical compositionscontaining them and various uses of the compounds.

The present invention provides the compounds of Formula (I) below or apharmaceutically acceptable salt or ester thereof:

wherein each of l, m, and n, independently is 0, 1, or 2, each of p, q,r, t, u, v, and w, independently is 0 or 1, and the sum of l, m, n, p,q, r, t, u, v, and w is 1, 2, or 3.

One subset of the compounds of Formula (I) includes those of Formula(IA):

Another subset of the compounds of Formula (I) includes those of Formula(IB):

The compounds of Formulae (I), (IA), and (IB) or pharmaceuticallyacceptable salts or esters thereof can include one or more of thefollowing features:

For example, the compounds or pharmaceutically acceptable salts oresters thereof each independently are in an isolated form, e.g., apurified form.

For example, the sum of l, m, n, p, q, r, t, u, v, and w is 1, namely,the compound of Formula (I) is a monohydroxylated compound (not countingthe two hydroxyls on the tetrahydrofuran ring) or a mono-N-oxidecompound. For example, 1 is 1, and each of m, n, p, q, r, t, u, v, andw, independently is 0. For example, m is 1, and each of l, n, p, q, r,t, u, v, and w, independently is 0. For example, n is 1, and each of l,m, p, q, r, t, u, v, and w, independently is 0. For example, u is 1, andeach of l, m, n, p, q, r, t, v, and w, independently is 0. For example,v is 1, and each of l, m, n, p, q, r, t, u, and w, independently is 0.For example, w is 1, and each of l, m, n, p, q, r, t, u, and v,independently is 0. For example, one of q, r, and t is 1.

For example, the sum of l, m, n, p, q, r, t, u, v, and w is 2, namely,the compound of Formula (I) is a di-hydroxylated compound (not countingthe two hydroxyls on the tetrahydrofuran ring), a di-N-oxide compound,or a mono-hydroxyl-mono-N-oxide compound (not counting the two hydroxylson the tetrahydrofuran ring).

For example, the sum of l, m, n, p, q, r, t, u, v, and w is 3, namely,the compound of Formula (I) is a tri-hydroxylated compound (not countingthe two hydroxyls on the tetrahydrofuran ring), a tri-N-oxide compound,a mono-hydroxyl-di-N-oxide compound (not counting the two hydroxyls onthe tetrahydrofuran ring), or a di-hydroxyl-mono-N-oxide compound (notcounting the two hydroxyls on the tetrahydrofuran ring).

In yet another aspect, the present invention features a compound ofFormula (II) below or a pharmaceutically acceptable salt or esterthereof:

wherein R₁ is unsubstituted t-butyl or t-butyl substituted with one ormore substituents selected from hydroxyl and oxo (i.e., ═O), R₂ is H,hydroxyl, unsubstituted i-propyl, or i-propyl substituted with one ormore hydroxyl, l′ is 0, 1, 2, or 3, each of m′ and n′, independently is0, 1, or 2, each of p′, q′, r′, t′, u′, v′, and w′, independently is 0or 1, and when R₁ is t-butyl substituted with only one hydroxyl, R₂ isH, hydroxyl, or i-propyl substituted with one or more hydroxyl, and whenR₁ is unsubstituted t-butyl, then (i) R₂ is hydroxyl, or i-propylsubstituted with one or more hydroxyl, or (ii) the sum of l′, m′, n′,p′, q′, r′, t′, u′, v′, and w′ is 1 or greater.

One subset of the compounds of Formula (II) includes those of Formula(IIA):

Another subset of the compounds of Formula (I) includes those of Formula(IIB):

The compounds of Formulae (II), (IIA), and (IIB) or pharmaceuticallyacceptable salts or esters thereof can include one or more of thefollowing features:

For example, the compounds or pharmaceutically acceptable salts oresters thereof each independently are in an isolated form, e.g., apurified form.

For example, the compound of Formula (II) is a mono-hydroxylatedcompound (not counting the two hydroxyls on the tetrahydrofuran ring).For example, R₁ is unsubstituted t-butyl, and (i) R₂ is hydroxyl ori-propyl substituted with one hydroxyl, and each of l′, m′, n′, u′, v′,and w′ is 0, or (ii) R₂ is H or unsubstituted i-propyl, one of l′, m′,n′, u′, v′, and w′ is 1 and the other five are each 0. For example, R₁is t-butyl substituted with only one hydroxyl, and R₂ is H, and each ofl′, m′, n′, u′, v′, and w′ is 0. For example, each of p′, q′, r′, andt′, independently is 0 or 1.

For example, the compound of Formula (II) is a carboxylic acid. Forexample, R₁ is t-butyl substituted with only one hydroxyl and one oxo,for example, the hydroxyl and oxo together with the carbon to which theyare attached form —COOH). For example, R₁ is —C(CH₃)₂COOH) and R₂ is Hor unsubstituted i-propyl, and each of l′, m′, n′, u′, v′, and w′ is 0.For example, each of p′, q′, r′, and t′, independently is 0 or 1.

For example, the compound of Formula (II) is a di-hydroxylated ortri-hydroxylated compound (not counting the two hydroxyls on thetetrahydrofuran ring). For example, R₁ is t-butyl substituted with onlyone hydroxyl, and (i) R₂ is hydroxyl or i-propyl substituted with one ortwo hydroxyl, and each of l′, m′, n′, u′, v′, and w′ is 0, or (ii) R₂ isH, hydroxyl, or i-propyl optionally substituted with one hydroxyl, oneof l′, m′, n′, u′, v′, and w′ is 1 and the other five are each 0. Forexample, R₁ is t-butyl substituted with two hydroxyl, and (i) R₂ is H,hydroxyl or i-propyl optionally substituted with one hydroxyl, and eachof l′, m′, n′, u′, v′, and w′ is 0, or (ii) R₂ is H, hydroxyl ori-propyl optionally substituted with one hydroxyl, one of l′, m′, n′,u′, v′, and w′ is 1 and the other five are each 0. For example, R₁ ist-butyl substituted with three hydroxyl, R₂ is H or unsubstitutedi-propyl, each of l′, m′, n′, u′, v′, and w′ is 0. For example, each ofp′, q′, r′, and t′, independently is 0 or 1.

For example, the compound of Formula (II) is a tetra-hydroxylatedcompound (not counting the two hydroxyls on the tetrahydrofuran ring).

For example, the compound of Formula (II) is a compound having five ormore hydroxyls (not including the two hydroxyls on the tetrahydrofuranring).

Unless otherwise specified, the term “Formula (I) or (II)” used herewithrefers to any of Formulae (I), (IA), (IB), (II), (IIA) and (IIB).

In one aspect, the present invention features a compound that isselected from those listed in Table 1, i.e., compounds 1-89, andpharmaceutically acceptable salts thereof.

In one aspect, the present invention features a compound that isselected from compounds 101-104, and 107-114 listed in Table 2, andpharmaceutically acceptable salts thereof.

This invention also provides a pharmaceutical composition comprising acompound of any of the Formulae described herein and a pharmaceuticallyacceptable carrier.

One aspect of the present invention is based in part upon the surprisingdiscovery that DOT1L inhibitors can effectively treat leukemia that ischaracterized by partial tandem duplication of the MLL gene. Anotheraspect of the present invention is based in part upon the surprisingdiscovery that DOT1L inhibitors can effectively treat leukemia that ischaracterized by overexpression of HOXA9, FLT3, MEIS1 and/or DOT1L.Specifically, tumors or tumor cells having increased mRNA or proteinlevel of at least one protein selected from the group consisting ofHOXA9, FLT3, MEIS1 and DOT1L are sensitive to the DOT1L inhibitors ofthe present invention. Accordingly, the present invention providesmethods of treating or alleviating a symptom of leukemia in a subject byadministering a therapeutically effective amount of a DOT1L inhibitordescribed herein to the subject, particular leukemia associated withoverexpression of at least one protein selected from the groupconsisting of HOXA9, FLT3, MEIS1 and DOT1L.

The compounds of the present invention inhibit the histonemethyltransferase activity of DOT1L or a mutant thereof. Based upon thesurprising discovery that methylation regulation by DOT1L involves intumor formation, particular tumors bearing an increased mRNA, proteinand/or activity (function) level of at least one protein selected fromthe group consisting of HOXA9, FLT3, MEIS1 and DOT1L, the compoundsdescribed herein are suitable candidates for treating cancers, i.e., todecrease methylation or restore methylation to roughly its level incounterpart normal cells.

The present invention features a method for treating or alleviating asymptom of cancer. The method includes administering to a subject inneed thereof, a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph, solvate, or stereoisomeror thereof.

The present invention provides methods for the treatment of a cancermediated by DOT1 (e.g., DOT1L)-mediated protein methylation in a subjectin need thereof by administering to a subject in need of such treatment,a therapeutically effective amount of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof. The present invention further provides theuse of a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, forthe preparation of a medicament useful for the treatment of a cancermediated by DOT1L-mediated protein methylation.

The present invention provides methods for the treatment of a cancer thecourse of which is influenced by modulating the methylation status ofhistones or other proteins, wherein said methylation status is mediatedat least in part by the activity of DOT1L. Modulation of the methylationstatus of histones can in turn influence the level of expression oftarget genes activated by methylation, and/or target genes suppressed bymethylation. The method includes administering to a subject in need ofsuch treatment, a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph, solvate, or stereoisomeror thereof.

The present invention also provides methods of protecting against orpreventing a cancer in which DOT1L-mediated protein methylation plays apart in a subject in need thereof by administering a therapeuticallyeffective amount of compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, to a subject in need of such treatment. The presentinvention also provides the use of compound of the present invention, ora pharmaceutically acceptable salt, prodrug, metabolite, polymorph,solvate, or stereoisomeror thereof, for the preparation of a medicamentuseful for the prevention of a cell proliferative disorder.

In one aspect, the cancer is a cancer selected from the group consistingof brain and CNS cancer, kidney cancer, ovarian cancer, pancreaticcancer, lung cancer, breast cancer, colon cancer, prostate cancer, or ahematological cancer. For example, the hematological cancer is leukemiaor lymphoma. Preferably the cancer is leukemia.

The present invention further provides the use of a compound of thepresent invention, or a pharmaceutically acceptable salt, ester,prodrug, metabolite, polymorph or solvate thereof in the treatment ofleukemia, or, for the preparation of a medicament useful for thetreatment of such leukemia. The leukemia can be acute or chronicleukemia. Preferably, the leukemia is acute myeloid leukemia, acutelymphocytic leukemia or mixed lineage leukemia. Exemplary leukemia thatmay be treated is mixed linage leukemia (MLL). Preferably, the MLL thatcan be treated by the compound of the present invention is chimericfusion of MLL, partial tandem duplication of the MLL gene (MLL-PTD) ornon-rearranged MLL.

Mixed lineage leukemia (MLL) is a genetically distinct form of acuteleukemia that constitutes over 70% of infant leukemias and approximately10% of adult acute myeloid leukemias (AML) (Hess, J. L. (2004), TrendsMol Med 10, 500-507; Krivtsov, A. V., and Armstrong, S. A. (2007), NatRev Cancer 7, 823-833). MLL represents a particularly aggressive form ofleukemia and patients with this disease generally have poor prognoses;these patients often suffer from early relapse after treatment withcurrent chemotherapies. There is thus a great and present need for newtreatment modalities for patients suffering with MLL.

A universal hallmark of MLL disease is a chromosomal translocationaffecting the MLL gene on chromosome 11q23 (Hess, 2004; Krivtsov andArmstrong, 2007). Normally, the MLL gene encodes for a SET-domainhistone methyltransferase that catalyzes the methylation of lysine 4 ofhistone H3 (H3K4) at specific gene loci (Milne et al. (2002) Mol Cell10, 1107-1117; Nakamura et al. (2002), Mol Cell 10, 1119-1128). Genelocalization is conferred by specific interactions with recognitionelements within MLL, external to the SET-domain (Ayton et al. (2004) MolCell Biol 24, 10470-10478; Slany et al., (1998) Mol Cell Biol 18,122-129; Zeleznik-Le et al. (1994) Proc Natl Acad Sci USA 91,10610-10614). In the disease-linked translocations, the catalyticSET-domain is lost and the remaining MLL protein is fused to a varietyof partners, including members of the AF and ENL family of proteins suchas AF4, AF9, AF10 and ENL (Hess, 2004; Krivtsov and Armstrong, 2007;Slany (2009) Haematologica 94, 984-993). These fusion partners arecapable of interacting directly, or indirectly, with another histonemethyltransferase, DOT1L (Bitoun et al. (2007) Hum Mol Genet 16, 92-106;Mohan et al. (2010) Genes Dev. 24, 574-589; Mueller et al. (2007) Blood110, 4445-4454; Mueller et al. (2009) PLoS Biol 7, e1000249; Okada etal. (2005) Cell 121, 167-178; Park et al. (2010) Protein J 29, 213-223;Yokoyama et al. (2010) Cancer Cell 17, 198-212; Zhang et al. (2006) JBiol Chem 281, 18059-18068). As a result, translocation products retaingene-specific recognition elements within the remainder of the MLLprotein, but also gain the ability to recruit DOT1L, to these locations(Monroe et al. (2010) Exp Hematol. 2010 Sep. 18. [Epub ahead of print]Pubmed PMID: 20854876; Mueller et al., 2007; Mueller et al., 2009; Okadaet al., 2005). DOT1L catalyzes the methylation of H3K79, a chromatinmodification associated with actively transcribed genes (Feng et al.(2002) Curr Biol 12, 1052-1058; Steger et al. (2008) Mol Cell Biol 28,2825-2839). The ectopic H3K79 methylation that results from MLL fusionprotein recruitment of DOT1L leads to enhanced expression ofleukemogenic genes, including HOXA9 and MEIS1 (Guenther et al. (2008)Genes & Development 22, 3403-3408; Krivtsov et al. (2008) Nat Rev Cancer7, 823-833; Milne et al. (2005) Cancer Res 65, 11367-11374; Monroe etal., 2010; Mueller et al., 2009; Okada et al., 2005; Thiel et al. (2010)Cancer Cell 17, 148-159). Hence, while DOT1L is not genetically alteredin the disease per se, its mislocated enzymatic activity is a directconsequence of the chromosomal translocation affecting MLL patients;thus, DOT1L has been proposed to be a catalytic driver of leukemogenesisin this disease (Krivtsov et al., 2008; Monroe et al., 2010; Okada etal., 2005; Yokoyama et al. (2010) Cancer Cell 17, 198-212). Furthersupport for a pathogenic role of DOT1L in MLL comes from studies inmodel systems that demonstrate a requirement for DOT1L in propagatingthe transforming activity of MLL fusion proteins (Mueller et al., 2007;Okada et al., 2005).

Evidence indicates that the enzymatic activity of DOT1L is critical topathogenesis in MLL and inhibition of DOT1L may provide a pharmacologicbasis for therapeutic intervention in this disease. Compound treatmentresults in selective, concentration-dependent killing of leukemia cellsbearing the MLL-translocation without effect on non-MLL transformedcells. Gene expression analysis of inhibitor treated cells showsdownregulation of genes aberrantly over expressed in MLL-rearrangedleukemias and similarities with gene expression changes caused bygenetic knockout of the DOT1L gene in a mouse model of MLL-AF9 leukemia.

MLL can be characterized by the genetic lesions of the MLL gene. Suchgenetic lesions include chromosomal rearrangements, such astranslocations, deletions, and/or duplications of the MLL gene. MLL hasbeen categorized or characterized as having a chimeric fusion of MLL,partial tandem duplication of the MLL gene (MLL-PTD), or nonrearrangedMLL. Chromosomal rearrangements or translocations can be identified bymethods known in the art. For example, chromosomal rearrangementsresulting in chimeric fusions can be detected by probe-based assays,such as FISH (fluorescence in situ hybridization) or sequenceamplification by PCR. Those chromosomal rearrangements that result inpartial tandem duplications are often difficult to detect by probe-basedassays, and therefore, other DNA sequencing methods known in the art maybe used, such as Sanger sequencing, de novo sequencing, shotgunsequencing, or next generation sequencing methods. MLL-PTD can beidentified by DNA sequencing. MLL chimeric fusions can be identified byFISH. Diagnosis of MLL can be performed by detection of rearrangementsof the MLL gene, or increased mRNA, protein, and/or activity level of atleast one protein selected from the group consisting of HOXA9, FLT3,MEIS1 and DOT1L, as further described herein.

Compounds of the present invention can selectively inhibit proliferationof tumor or tumor cells characterized with an increased mRNA, proteinand/or activity (function) level of at least one protein selected fromthe group consisting of HOXA9, FLT3, MEIS1 and DOT1L.

Accordingly, the present invention provides methods for treating oralleviating a symptom of leukemia characterized with an increased mRNA,protein and/or activity (function) level of at least one proteinselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L by acompound of the present invention, or a pharmaceutically acceptablesalt, ester, prodrug, metabolite, polymorph or solvate thereof.Exemplary leukemia that may be treated is mixed linage leukemia (MLL).Preferably, MLL that can be treated by the compound of the presentinvention is chimeric fusion of MLL, partial tandem duplication of MLL(MLL-PTD) or nonrearranged MLL.

The present invention also provides methods for treating or alleviatinga symptom of leukemia characterized by the presence of a genetic lesionof MLL. For example, this method comprises obtaining sample from thesubject; detecting the presence of a genetic lesion of MLL in thesample; and when the genetic lesion is present in the sample,administering to the subject a therapeutically effective amount of aDOT1L inhibitor (i.e., a compound selected from compounds 1-89, 101-104,and 107-114 and pharmaceutically acceptable salts thereof). The geneticlesion is chimeric fusion of MLL or MLL-PTD.

The present invention also provides methods for treating a disordermedicated by translocation, deletion and/or duplication of a gene onchromosome 11q23, comprising administering to a subject in need thereofa therapeutically effective amount of a compound selected from compounds1-89, 101-104, and 107-114 and pharmaceutically acceptable saltsthereof.

In other aspects, the present invention provides personalized medicine,treatment and/or cancer management for a subject by genetic screening ofincreased gene expression (mRNA or protein), and/or increased functionor activity level of at least one protein selected from the groupconsisting of HOXA9, FLT3, MEIS1 and DOT1L in the subject. For example,the present invention provides methods for treating, preventing oralleviating a symptom of cancer or a precancerous condition bydetermining responsiveness of the subject to a DOT1L inhibitor and whenthe subject is responsive to the DOT1L inhibitor, administering to thesubject a therapeutically effective amount of the DOT1L inhibitor, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph,solvate, or stereoisomeror thereof. The responsiveness is determined byobtaining a sample from the subject and detecting increased mRNA orprotein, and/or increased activity level of at least one proteinselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L, andthe presence of such gain of expression and/or function indicates thatthe subject is responsive to the DOT1L inhibitor. Once theresponsiveness of a subject is determined, a therapeutically effectiveamount of a DOT1L inhibitor, for example, any compound selected fromcompounds 1-89, 101-104, and 107-114 and pharmaceutically acceptablesalts thereof, can be administered. The therapeutically effective amountof a DOT1L inhibitor can be determined by one of ordinary skill in theart.

As used herein, the term “responsiveness” is interchangeable with terms“responsive”, “sensitive”, and “sensitivity”, and it is meant that asubject is showing therapeutic responses when administered an DOT1Linhibitor, e.g., tumor cells or tumor tissues of the subject undergoapoptosis and/or necrosis, and/or display reduced growing, dividing, orproliferation. This term is also meant that a subject will or has ahigher probability, relative to the population at large, of showingtherapeutic responses when administered an DOT1L inhibitor, e.g., tumorcells or tumor tissues of the subject undergo apoptosis and/or necrosis,and/or display reduced growing, dividing, or proliferation.

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which refers to a subject having a disorder in whichDOT1L-mediated protein methylation plays a part, or a subject having anincreased risk of developing such disorder relative to the population atlarge. A subject in need thereof may be a subject having a disorderassociated DOT1L. A subject in need thereof can have a precancerouscondition. Preferably, a subject in need thereof has cancer. A subjectin need thereof can have cancer associated with DOT1L. A subject in needthereof can have cancer associated with increased expression (mRNA orprotein) and/or activity level of at least one protein selected from thegroup consisting of HOXA9, FLT3, MEIS1 and DOT1L. In a preferred aspect,a subject in need thereof has one or more cancers selected from thegroup consisting of brain and central nervous system (CNS) cancer, headand neck cancer, kidney cancer, ovarian cancer, pancreatic cancer,leukemia, lung cancer, lymphoma, myeloma, sarcoma, breast cancer,prostate cancer and a hematological cancer. Preferably, a subject inneed thereof has a hematologic cancer, wherein the hematologic cancer isleukemia or lymphoma. Exemplary leukemia is MLL. Other hematologiccancers of the present invention can include multiple myeloma, lymphoma(including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhoodlymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia(including childhood leukemia, hairy-cell leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, chronic lymphocytic leukemia,chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cellleukemia), myeloid neoplasms and mast cell neoplasms.

As used herein, a “subject” includes a mammal. The mammal can be e.g., ahuman or appropriate non-human mammal, such as primate, mouse, rat, dog,cat, cow, horse, goat, camel, sheep or a pig. The subject can also be abird or fowl. In one embodiment, the mammal is a human. A subject can bemale or female.

A subject in need thereof can be one who has been previously diagnosedor identified as having cancer or a precancerous condition. A subject inneed thereof can also be one who is having (suffering from) cancer or aprecancerous condition. Alternatively, a subject in need thereof can beone who is having an increased risk of developing such disorder relativeto the population at large (i.e., a subject who is predisposed todeveloping such disorder relative to the population at large).

Optionally a subject in need thereof has already undergone, isundergoing or will undergo, at least one therapeutic intervention forthe cancer or precancerous condition.

A subject in need thereof may have refractory cancer on most recenttherapy. “Refractory cancer” means cancer that does not respond totreatment. The cancer may be resistant at the beginning of treatment orit may become resistant during treatment. Refractory cancer is alsocalled resistant cancer. In some embodiments, the subject in needthereof has cancer recurrence following remission on most recenttherapy. In some embodiments, the subject in need thereof received andfailed all known effective therapies for cancer treatment. In someembodiments, the subject in need thereof received at least one priortherapy.

In some embodiments, a subject in need thereof may have a secondarycancer as a result of a previous therapy. “Secondary cancer” meanscancer that arises due to or as a result from previous carcinogenictherapies, such as chemotherapy. In some embodiments, the secondarycancer is a hematologic cancer, such as leukemia.

In any method of the present invention, a subject in need thereof mayhave increased mRNA, protein, and/or activity level of at least of atleast one signaling component downstream of at least one proteinselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L. Suchdownstream components are readily known in the art, and can includeother transcription factors, or signaling proteins.

As used herein, the term “increase in activity” refers to increased or again of function of a gene product/protein compared to the wild type. Inone aspect of the present invention, increased activity can be caused byincreased mRNA and/or increased protein levels.

Increased mRNA levels can be caused by gene amplification and increasedtranscription, for example. Increased protein levels can be caused byincreased stability, inhibition of degradation pathways, or increasedtranscription. Alternatively, increased activity levels can be caused bya gain of function mutation resulting from a point mutation (e.g., asubstitution, a missense mutation, or a nonsense mutation), aninsertion, and/or a deletion, or a rearrangement in a polypeptideselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L, or anucleic acid sequence encoding a polypeptide selected from the groupconsisting of HOXA9, FLT3, MEIS1 and DOT1L. The mutations referredherein are somatic mutations. The term “somatic mutation” refers to adeleterious alteration in at least one gene allele that is not found inevery cell of the body, but is found only in isolated cells. Acharacteristic of the somatic mutations as used herein is, that they arerestricted to particular tissues or even parts of tissues or cellswithin a tissue and are not present in the whole organism harboring thetissues or cells. The term “wild-type” refers to a gene or gene productthat has the characteristics of that gene or gene product when isolatedfrom a naturally occurring source. A wild-type gene is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

Accordingly, an increase in mRNA or protein expression and/or activitylevels can be detected using any suitable method available in the art.For example, an increase in activity level can be detected by measuringthe biological function of a gene product, such as the histonemethyltransferase activity of DOT1L (i.e., methylation of histonesubstrates such as H3K79 by immunoblot); transcriptional activity ofHOXA9 or MEIS1 (i.e., expression levels of HOXA9 or MEIS1 target genesby RT-PCR); or phosphorylation activity of FLT3 (i.e., phosphorylationstatus of FLT3 targets by immunoblot or radioimmunoassay).Alternatively, a gain of function mutation can be determined bydetecting any alternation in a nucleic acid sequence encoding a proteinselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L. Forexample, a nucleic acid sequence encoding HOXA9, FLT3, MEIS1 and DOT1Lhaving a gain of function mutation can be detected by whole-genomeresequencing or target region resequencing (the latter also known astargeted resequencing) using suitably selected sources of DNA andpolymerase chain reaction (PCR) primers in accordance with methods wellknown in the art. The method typically and generally entails the stepsof genomic DNA purification, PCR amplification to amplify the region ofinterest, cycle sequencing, sequencing reaction cleanup, capillaryelectrophoresis, and/or data analysis. Alternatively or in addition, themethod may include the use of microarray-based targeted region genomicDNA capture and/or sequencing. Kits, reagents, and methods for selectingappropriate PCR primers and performing resequencing are commerciallyavailable, for example, from Applied Biosystems, Agilent, and NimbleGen(Roche Diagnostics GmbH). Detection of mRNA expression can be detectedby methods known in the art, such as Northern blot, nucleic acid PCR,and quantitative RT-PCR. Detection of polypeptide expression (i.e.,wild-type or mutant) can be carried out with any suitable immunoassay inthe art, such as Western blot analysis.

By “sample” it means any biological sample derived from the subject,includes but is not limited to, cells, tissues samples, body fluids(including, but not limited to, mucus, blood, plasma, serum, urine,saliva, and semen), tumor cells, and tumor tissues. Preferably, thesample is selected from bone marrow, peripheral blood cells, blood,plasma and serum. Samples can be provided by the subject under treatmentor testing. Alternatively samples can be obtained by the physicianaccording to routine practice in the art.

The present invention also provides methods for diagnosing leukemia in asubject by obtaining a sample from the subject and detecting anincreased mRNA, protein and/or activity level of at least one proteinselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L, andthe presence of such increased mRNA, protein and/or activity levelindicates that the subject has or is at risk for developing leukemiacompared to a subject without such increased mRNA, protein and/oractivity level, or a subject that does not have leukemia.

The present invention also provides methods for determiningpredisposition of a subject to leukemia by obtaining a sample from thesubject and detecting an increased mRNA, protein and/or activity levelof at least one protein selected from the group consisting of HOXA9,FLT3, MEIS1 and DOT1L, and the presence of such increased mRNA, proteinand/or activity level indicates that the subject is predisposed to(i.e., having higher risk of) developing leukemia compared to a subjectwithout such increased mRNA, protein and/or activity level.

The term “predisposed” as used herein in relation to cancer or aprecancerous condition is to be understood to mean the increasedprobability (e.g., at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, or more increase in probability) that asubject with an increased mRNA, protein and/or activity level of atleast one protein selected from the group consisting of HOXA9, FLT3,MEIS1 and DOT1L, will suffer leukemia, as compared to the probabilitythat another subject not having an increased mRNA, protein and/oractivity level of at least one protein selected from the groupconsisting of HOXA9, FLT3, MEIS1 and DOT1L, will suffer leukemia, undercircumstances where other risk factors (e.g., chemical/environment,food, and smoking history, etc.) for having leukemia between thesubjects are the same.

“Risk” in the context of the present invention, relates to theprobability that an event will occur over a specific time period and canmean a subject's “absolute” risk or “relative” risk. Absolute risk canbe measured with reference to either actual observation post-measurementfor the relevant time cohort, or with reference to index valuesdeveloped from statistically valid historical cohorts that have beenfollowed for the relevant time period. Relative risk refers to the ratioof absolute risks of a subject compared either to the absolute risks oflow risk cohorts or an average population risk, which can vary by howclinical risk factors are assessed. Odds ratios, the proportion ofpositive events to negative events for a given test result, are alsocommonly used (odds are according to the formula p/(1−p) where p is theprobability of event and (1−p) is the probability of no event) tono-conversion.

In other example, the present invention provides methods of cancermanagement in a subject by determining predisposition of the subject toa cancer or a precancerous condition periodically. The methods comprisesteps of obtaining a sample from the subject and detecting increasedmRNA or protein, and/or increased activity level of at least one proteinselected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L, andthe presence of such gain of expression and/or function indicates thatthe subject is predisposed to developing the cancer or the precancerouscondition compared to a subject without such gain of mRNA or proteinexpression and/or function of the at least one protein selected from thegroup consisting of HOXA9, FLT3, MEIS1 and DOT1L.

Any compounds (e.g., DOT1L inhibitor) of the present invention can beused for the methods described above.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue.

A cell proliferative disorder includes a precancer or a precancerouscondition. A cell proliferative disorder includes cancer. Preferably,the methods provided herein are used to treat or alleviate a symptom ofcancer. The term “cancer” includes solid tumors, as well as, hematologictumors and/or malignancies. A “precancer cell” or “precancerous cell” isa cell manifesting a cell proliferative disorder that is a precancer ora precancerous condition. A “cancer cell” or “cancerous cell” is a cellmanifesting a cell proliferative disorder that is a cancer. Anyreproducible means of measurement may be used to identify cancer cellsor precancerous cells. Cancer cells or precancerous cells can beidentified by histological typing or grading of a tissue sample (e.g., abiopsy sample). Cancer cells or precancerous cells can be identifiedthrough the use of appropriate molecular markers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, urinarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodeimaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute lymphocyticleukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer,liver cancer, lung cancer, non-small cell lung cancer, small cell lungcancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary centralnervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma,melanoma, intraocular (eye) melanoma, merkel cell carcinoma,mesothelioma malignant, mesothelioma, metastatic squamous neck cancer,mouth cancer, cancer of the tongue, multiple endocrine neoplasiasyndrome, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, chronic myelogenousleukemia, acute myeloid leukemia, multiple myeloma, chronicmyeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oralcancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer,ovarian epithelial cancer, ovarian low malignant potential tumor,pancreatic cancer, islet cell pancreatic cancer, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiplemyeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renalpelvis and ureter, transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors,Kaposi Sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma,skin cancer (non-melanoma), skin cancer (melanoma), merkel cell skincarcinoma, small intestine cancer, soft tissue sarcoma, squamous cellcarcinoma, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, testicular cancer, throat cancer, thymoma,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancerof the renal pelvis and ureter and other urinary organs, gestationaltrophoblastic tumor, urethral cancer, endometrial uterine cancer,uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer,and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. Preferably,compositions of the present invention may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent invention or a hematologic cell proliferative disorder of thepresent invention. A hematologic cancer of the present invention caninclude multiple myeloma, lymphoma (including Hodgkin's lymphoma,non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas oflymphocytic and cutaneous origin), leukemia (including childhoodleukemia, hairy-cell leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloidneoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. Preferably, compositions of the present invention may be usedto treat lung cancer or cell proliferative disorders of the lung. Lungcancer can include all forms of cancer of the lung. Lung cancer caninclude malignant lung neoplasms, carcinoma in situ, typical carcinoidtumors, and atypical carcinoid tumors. Lung cancer can include smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lungcancer can include “scar carcinoma,” bronchioalveolar carcinoma, giantcell carcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. Lung cancer can include lung neoplasms having histologic andultrastructual heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. Preferably,compositions of the present invention may be used to treat colon canceror cell proliferative disorders of the colon. Colon cancer can includeall forms of cancer of the colon. Colon cancer can include sporadic andhereditary colon cancers. Colon cancer can include malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. Compositions of the present invention may beused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of NO; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer.Preferably, compositions of the present invention may be used to treatbreast cancer. Breast cancer includes all forms of cancer of the breast.Breast cancer can include primary epithelial breast cancers. Breastcancer can include cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. Breast cancer can includecarcinoma of the breast, ductal carcinoma of the breast, lobularcarcinoma of the breast, undifferentiated carcinoma of the breast,cystosarcoma phyllodes of the breast, angiosarcoma of the breast, andprimary lymphoma of the breast. Breast cancer can include Stage I, II,IIIA, IIIB, IIIC and IV breast cancer. Ductal carcinoma of the breastcan include invasive carcinoma, invasive carcinoma in situ withpredominant intraductal component, inflammatory breast cancer, and aductal carcinoma of the breast with a histologic type selected from thegroup consisting of comedo, mucinous (colloid), medullary, medullarywith lymphcytic infiltrate, papillary, scirrhous, and tubular. Lobularcarcinoma of the breast can include invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. Breast cancer can include Paget'sdisease, Paget's disease with intraductal carcinoma, and Paget's diseasewith invasive ductal carcinoma. Breast cancer can include breastneoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

Preferably, compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph, or solvate thereof, maybe used to treat breast cancer. A breast cancer that is to be treatedcan include familial breast cancer. A breast cancer that is to betreated can include sporadic breast cancer. A breast cancer that is tobe treated can arise in a male subject. A breast cancer that is to betreated can arise in a female subject. A breast cancer that is to betreated can arise in a premenopausal female subject or a postmenopausalfemale subject. A breast cancer that is to be treated can arise in asubject equal to or older than 30 years old, or a subject younger than30 years old. A breast cancer that is to be treated has arisen in asubject equal to or older than 50 years old, or a subject younger than50 years old. A breast cancer that is to be treated can arise in asubject equal to or older than 70 years old, or a subject younger than70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich, or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, may be used totreat or prevent a cell proliferative disorder of the breast, or totreat or prevent breast cancer, in a subject having an increased risk ofdeveloping breast cancer relative to the population at large. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female subject with a family history orpersonal history of breast cancer. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalesubject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, orboth. A subject with an increased risk of developing breast cancerrelative to the population at large is a female subject with a familyhistory of breast cancer and a germ-line or spontaneous mutation inBRCA1 or BRCA2, or both. A subject with an increased risk of developingbreast cancer relative to the population at large is a female who isgreater than 30 years old, greater than 40 years old, greater than 50years old, greater than 60 years old, greater than 70 years old, greaterthan 80 years old, or greater than 90 years old. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a subject with atypical hyperplasia of the breast, ductalcarcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma insitu (LCIS), lobular neoplasia, or a stage 0 growth or lesion of thebreast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, that has been or will be tested in one ormore in vitro or in vivo biological assays, in order to determine ifthat compound is likely to elicit a desired biological or medicalresponse in a cell, tissue, system, animal or human that is being soughtby a researcher or clinician. A candidate compound is a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof. The biological or medicalresponse can be the treatment of cancer. The biological or medicalresponse can be treatment or prevention of a cell proliferativedisorder. In vitro or in vivo biological assays can include, but are notlimited to, enzymatic activity assays, electrophoretic mobility shiftassays, reporter gene assays, in vitro cell viability assays, and theassays described herein.

For example, an in vitro biological assay that can be used includes thesteps of (1) mixing a histone substrate (e.g., an isolated histonesample for a histone or modified histone of interest, or an isolatedoligonucleosome substrate) with recombinant DOT1L enzyme (e.g.,recombinant protein containing amino acids 1-416); (2) adding acandidate compound of the invention to this mixture; (3) addingnon-radioactive and ³H-labeled S-Adenosyl methionine (SAM) to start thereaction; (4) adding excessive amount of non-radioactive SAM to stop thereaction; (4) washing off the free non-incorporated ³H-SAM; and (5)detecting the quantity of ³H-labeled histone substrate by any methodsknown in the art (e.g., by a PerkinElmer TopCount platereader).

For example, an in vitro cell viability assay that can be used includesthe steps of (1) culturing cells (e.g., EOL-1 cells) in the presence ofincreasing concentration of a candidate compound; (2) determining viablecell number every 3-4 days by methods known in the art (e.g., using theMillipore Guava Viacount assay); (3) plotting concentration-dependencegrowth curves; and optionally (4) calculating IC₅₀ values from theconcentration-dependence growth curves using methods known in the art(e.g., using GraphPad Prism Software).

For example, a histone methylation assay that can be used includes thesteps of (1) culturing cells (e.g., EOL-1 cells) in the presence of acandidate compound; (2) harvesting the cells; (3) extracting histoneproteins, using methods known in the art (e.g., sulfuric acidprecipitation); (4) fractionating histone extracts by SDS-PAGEelectrophoresis and transferring to a filter; (5) probing the filterwith antibodies specific to a protein or methylated-protein of interest(e.g., H3K79me2-specific antibody and total histone H3-specificantibody); and (6) detecting the signal of the antibodies using methodsknown in the art (e.g., Li-cor Odyssey infrared imager).

For example, a gene expression assay that can be used includes the stepsof (1) culturing cells (e.g., EOL-1, Molm13, MV411, LOUCY, SemK2, Reh,HL60, BV173, or Jurkat cells) in the presence or absence of a candidatecompound; (2) harvesting the cells; (3) extracting the RNA using methodsknown in the art (e.g., Qiagen RNeasy Kit); (4) synthesizing cDNA fromthe extracted RNA (e.g., Applied Biosystems reverse transcriptase kit);(5) preparing qPCR reactions using, for example, primers and probes(e.g., predesigned labeled primer and probe sets for HOXA9, MEIS1, FLT3,DOT1L, and β2-microglobulin from Applied Biosystems), synthesized samplecDNA, and qPCR master mix reagent (e.g., Applied Biosystems Taqmanuniversal PCR master mix); (6) running samples on PCR machine (e.g.,Applied Biosystems); (7) analysis of the data and calculation ofrelative gene expression.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of a single active compound. For example, cancer monotherapy withone of the compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof, to asubject in need of treatment of cancer. In one aspect, the single activecompound is a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can also beused to prevent a disease, condition or disorder. As used herein,“preventing” or “prevent” describes reducing or eliminating the onset ofthe symptoms or complications of the disease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

In another aspect of the invention, severity describes the degree towhich a tumor has secreted growth factors, degraded the extracellularmatrix, become vascularized, lost adhesion to juxtaposed tissues, ormetastasized. Moreover, severity describes the number of locations towhich a primary tumor has metastasized. Finally, severity includes thedifficulty of treating tumors of varying types and locations. Forexample, inoperable tumors, those cancers which have greater access tomultiple body systems (hematological and immunological tumors), andthose which are the most resistant to traditional treatments areconsidered most severe. In these situations, prolonging the lifeexpectancy of the subject and/or reducing pain, decreasing theproportion of cancerous cells or restricting cells to one system, andimproving cancer stage/tumor grade/histological grade/nuclear grade areconsidered alleviating a sign or symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

As a cancer grows, it begins to push on nearby organs, blood vessels,and nerves. This pressure creates some of the signs and symptoms ofcancer. If the cancer is in a critical area, such as certain parts ofthe brain, even the smallest tumor can cause early symptoms.

But sometimes cancers start in places where it does not cause anysymptoms until the cancer has grown quite large. Pancreas cancers, forexample, do not usually grow large enough to be felt from the outside ofthe body. Some pancreatic cancers do not cause symptoms until they beginto grow around nearby nerves (this causes a backache). Others growaround the bile duct, which blocks the flow of bile and leads to ayellowing of the skin known as jaundice. By the time a pancreatic cancercauses these signs or symptoms, it has usually reached an advancedstage.

A cancer may also cause symptoms such as fever, fatigue, or weight loss.This may be because cancer cells use up much of the body's energy supplyor release substances that change the body's metabolism. Or the cancermay cause the immune system to react in ways that produce thesesymptoms.

Sometimes, cancer cells release substances into the bloodstream thatcause symptoms not usually thought to result from cancers. For example,some cancers of the pancreas can release substances which cause bloodclots to develop in veins of the legs. Some lung cancers makehormone-like substances that affect blood calcium levels, affectingnerves and muscles and causing weakness and dizziness

Cancer presents several general signs or symptoms that occur when avariety of subtypes of cancer cells are present. Most people with cancerwill lose weight at some time with their disease. An unexplained(unintentional) weight loss of 10 pounds or more may be the first signof cancer, particularly cancers of the pancreas, stomach, esophagus, orlung.

Fever is very common with cancer, but is more often seen in advanceddisease. Almost all patients with cancer will have fever at some time,especially if the cancer or its treatment affects the immune system andmakes it harder for the body to fight infection. Less often, fever maybe an early sign of cancer, such as with leukemia or lymphoma.

Fatigue may be an important symptom as cancer progresses. It may happenearly, though, in cancers such as with leukemia, or if the cancer iscausing an ongoing loss of blood, as in some colon or stomach cancers.

Pain may be an early symptom with some cancers such as bone cancers ortesticular cancer. But most often pain is a symptom of advanced disease.

Along with cancers of the skin (see next section), some internal cancerscan cause skin signs that can be seen. These changes include the skinlooking darker (hyperpigmentation), yellow (jaundice), or red(erythema); itching; or excessive hair growth.

Alternatively, or in addition, cancer subtypes present specific signs orsymptoms. Changes in bowel habits or bladder function could indicatecancer. Long-term constipation, diarrhea, or a change in the size of thestool may be a sign of colon cancer. Pain with urination, blood in theurine, or a change in bladder function (such as more frequent or lessfrequent urination) could be related to bladder or prostate cancer.

Changes in skin condition or appearance of a new skin condition couldindicate cancer. Skin cancers may bleed and look like sores that do notheal. A long-lasting sore in the mouth could be an oral cancer,especially in patients who smoke, chew tobacco, or frequently drinkalcohol. Sores on the penis or vagina may either be signs of infectionor an early cancer.

Unusual bleeding or discharge could indicate cancer. Unusual bleedingcan happen in either early or advanced cancer. Blood in the sputum(phlegm) may be a sign of lung cancer. Blood in the stool (or a dark orblack stool) could be a sign of colon or rectal cancer. Cancer of thecervix or the endometrium (lining of the uterus) can cause vaginalbleeding. Blood in the urine may be a sign of bladder or kidney cancer.A bloody discharge from the nipple may be a sign of breast cancer.

A thickening or lump in the breast or in other parts of the body couldindicate the presence of a cancer. Many cancers can be felt through theskin, mostly in the breast, testicle, lymph nodes (glands), and the softtissues of the body. A lump or thickening may be an early or late signof cancer. Any lump or thickening could be indicative of cancer,especially if the formation is new or has grown in size.

Indigestion or trouble swallowing could indicate cancer. While thesesymptoms commonly have other causes, indigestion or swallowing problemsmay be a sign of cancer of the esophagus, stomach, or pharynx (throat).

Recent changes in a wart or mole could be indicative of cancer. Anywart, mole, or freckle that changes in color, size, or shape, or losesits definite borders indicates the potential development of cancer. Forexample, the skin lesion may be a melanoma.

A persistent cough or hoarseness could be indicative of cancer. A coughthat does not go away may be a sign of lung cancer. Hoarseness can be asign of cancer of the larynx (voice box) or thyroid.

While the signs and symptoms listed above are the more common ones seenwith cancer, there are many others that are less common and are notlisted here. However, all art-recognized signs and symptoms of cancerare contemplated and encompassed by the instant invention.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively on a canceror precancerous cell but not on a normal cell. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively to modulateone molecular target (e.g., a target protein methyltransferase) but doesnot significantly modulate another molecular target (e.g., a non-targetprotein methyltransferase). The invention also provides a method forselectively inhibiting the activity of an enzyme, such as a proteinmethyltransferase. Preferably, an event occurs selectively in populationA relative to population B if it occurs greater than two times morefrequently in population A as compared to population B. An event occursselectively if it occurs greater than five times more frequently inpopulation A. An event occurs selectively if it occurs greater than tentimes more frequently in population A; more preferably, greater thanfifty times; even more preferably, greater than 100 times; and mostpreferably, greater than 1000 times more frequently in population A ascompared to population B. For example, cell death would be said to occurselectively in cancer cells if it occurred greater than twice asfrequently in cancer cells as compared to normal cells.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can modulatethe activity of a molecular target (e.g., a target proteinmethyltransferase). Modulating refers to stimulating or inhibiting anactivity of a molecular target. Preferably, a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, modulates the activity of a moleculartarget if it stimulates or inhibits the activity of the molecular targetby at least 2-fold relative to the activity of the molecular targetunder the same conditions but lacking only the presence of saidcompound. More preferably, a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, modulates the activity of a molecular target if itstimulates or inhibits the activity of the molecular target by at least5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least100-fold relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, does notsignificantly modulate the activity of a molecular target if theaddition of the compound does not stimulate or inhibit the activity ofthe molecular target by greater than 10% relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, demonstrates a minimum of afourfold differential, preferably a tenfold differential, morepreferably a fifty fold differential, in the dosage required to achievea biological effect. Preferably, a compound of the present invention, ora pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, demonstrates this differential across the range ofinhibition, and the differential is exemplified at the IC₅₀, i.e., a 50%inhibition, for a molecular target of interest.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof can result in modulation (i.e.,stimulation or inhibition) of an activity of a protein methyltransferaseof interest.

The present invention provides methods to assess biological activity ofa compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof or methods ofidentifying a test compound as a modulator (e.g., an inhibitor) ofDOT1L. DOT1L polypeptides and nucleic acids can be used to screen forcompounds that bind to and/or modulate (e.g., increase or decrease) oneor more biological activities of DOT1L, including but not limited toH3K79 HMTase activity, SAM binding activity, histone and/or nucleosomebinding activity, AF10 binding activity, AF10-MLL or other MLL fusionprotein binding activity, and/or any other biological activity ofinterest. A DOT1L polypeptide can be a functional fragment of afull-length DOT1L polypeptide or functional equivalent thereof, and maycomprise any DOT1 domain of interest, including but not limited to thecatalytic domain, the SAM binding domain and/or the positively chargeddomain, the AF10 interaction domain and/or a nuclear export signal.

Methods of assessing DOT1L binding to histones, nucleosomes, nucleicacids or polypeptides can be carried out using standard techniques thatwill be apparent to those skilled in the art (see the Exemplificationfor exemplary methods). Such methods include yeast and mammaliantwo-hybrid assays and co-immunoprecipitation techniques.

For example, a compound that modulates DOT1L H3K79 HMTase activity canbe verified by: contacting a DOT1L polypeptide with a histone or peptidesubstrate comprising H3 in the presence of a test compound; detectingthe level of H3K79 methylation of the histone or peptide substrate underconditions sufficient to provide H3K79 methylation, wherein an elevationor reduction in H3K79 methylation in the presence of the test compoundas compared with the level of histone H3K79 methylation in the absenceof the test compound indicates that the test compound modulates DOT1LH3K79 HMTase activity.

The screening methods of the invention can be carried out in acell-based or cell-free system. As a further alternative, the assay canbe performed in a whole animal (including transgenic non-human animals).Further, with respect to cell-based systems, the DOT1L polypeptide (orany other polypeptide used in the assay) can be added directly to thecell or can be produced from a nucleic acid in the cell. The nucleicacid can be endogenous to the cell or can be foreign (e.g., agenetically modified cell).

In some assays, immunological reagents, e.g., antibodies and antigens,are employed. Fluorescence can be utilized in the measurement ofenzymatic activity in some assays. As used herein, “fluorescence” refersto a process through which a molecule emits a photon as a result ofabsorbing an incoming photon of higher energy by the same molecule.Specific methods for assessing the biological activity of the disclosedcompounds are described in the examples.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof results in modulation (i.e.,stimulation or inhibition) of an activity of an intracellular target(e.g., substrate). Several intracellular targets can be modulated withthe compounds of the present invention, including, but not limited to,protein methyltrasferase.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

As used herein, “a cell cycle checkpoint pathway” refers to abiochemical pathway that is involved in modulation of a cell cyclecheckpoint. A cell cycle checkpoint pathway may have stimulatory orinhibitory effects, or both, on one or more functions comprising a cellcycle checkpoint. A cell cycle checkpoint pathway is comprised of atleast two compositions of matter, preferably proteins, both of whichcontribute to modulation of a cell cycle checkpoint. A cell cyclecheckpoint pathway may be activated through an activation of one or moremembers of the cell cycle checkpoint pathway. Preferably, a cell cyclecheckpoint pathway is a biochemical signaling pathway.

As used herein, “cell cycle checkpoint regulator” refers to acomposition of matter that can function, at least in part, in modulationof a cell cycle checkpoint. A cell cycle checkpoint regulator may havestimulatory or inhibitory effects, or both, on one or more functionscomprising a cell cycle checkpoint. A cell cycle checkpoint regulatorcan be a protein or not a protein.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, is not significantly cytotoxic to normal cells. Atherapeutically effective amount of a compound is not significantlycytotoxic to normal cells if administration of the compound in atherapeutically effective amount does not induce cell death in greaterthan 10% of normal cells. A therapeutically effective amount of acompound does not significantly affect the viability of normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. In anaspect, cell death occurs by apoptosis.

Contacting a cell with a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, can induce or activate cell death selectively in cancercells. Administering to a subject in need thereof a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce or activate celldeath selectively in cancer cells. Contacting a cell with a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce cell deathselectively in one or more cells affected by a cell proliferativedisorder. Preferably, administering to a subject in need thereof acompound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, induces celldeath selectively in one or more cells affected by a cell proliferativedisorder.

The present invention relates to a method of treating or preventingcancer by administering a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, to a subject in need thereof, where administration ofthe compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, results in oneor more of the following: accumulation of cells in Gi and/or S phase ofthe cell cycle, cytotoxicity via cell death in cancer cells without asignificant amount of cell death in normal cells, antitumor activity inanimals with a therapeutic index of at least 2, and activation of a cellcycle checkpoint. As used herein, “therapeutic index” is the maximumtolerated dose divided by the efficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention

The compounds of the instant invention can also be utilized to treat orprevent cancer either in monotherapy or combination therapy.Accordingly, in one aspect, the present disclosure also provides acomposition comprising a compound described herein, or apharmaceutically acceptable salt thereof, and one or more therapeuticagents. The present invention provides for the administration of acompound described herein, or a pharmaceutically acceptable saltthereof, and one or more therapeutic agents as a co-formulation orseparate formulations, wherein the administration of formulations issimultaneous, sequential, or in alternation. In one embodiment, the oneor more therapeutic agents can be an agent that is recognized in the artas being useful to treat the disease or condition being treated by thecomposition of the present invention. In another embodiment, the one ormore therapeutic agents can be an agent that is not recognized in theart as being useful to treat the disease or condition being treated bythe composition of the present invention. In one aspect, the othertherapeutic agents can be an agent that imparts a beneficial attributeto the composition of the present invention (e.g., an agent that affectsthe viscosity of the composition). The beneficial attribute to thecomposition of the present invention includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of a compound of Formula (I) or (II) and one or moretherapeutic agents. For example, the one or more therapeutic agents canbe anticancer agents or chemotherapeutic agents.

For example, the one or more therapeutic agents can be selected fromAra-C, Daunorubicin, Decitabine, Vidaza, Mitoxantrone, JQ1, IBET151,Panobinostat, Vorinostat, Quizartinib, Midostaurin, Tranylcypromine,LSD1 inhibitor II, Navitoclax, or functional analogs, derivatives,prodrugs, and metabolites thereof. Preferably, the therapeutic agent isAra-C or Daunorubicin or functional analogs, derivatives, prodrugs, andmetabolites thereof. For example, a composition of the inventioncomprises Ara-C and a compound of Formula (I) or (II), or apharmaceutically acceptable salt thereof. For example, Ara-C and acompound of Formula (I) or (II), or a pharmaceutically acceptable saltthereof are administered together or separately, simultaneously orsequentially, or in alternation.

In some embodiments, the therapeutic agents are topoisomerase inhibitors(e.g., Mitoxantrone), hypomethylating agents (e.g., Decitabine orVidaza), Bromodomain inhibitors (e.g., IBET-151), HDAC inhibitors (e.g.,Panobinostat), Bcl-2 inhibitors (e.g., Navitoclax) or FLT inhibitors(e.g., Quizartinib).

In one embodiment, the other therapeutic agent is an anticancer agent.In one embodiment, the anticancer agent is a compound that affectshistone modifications, such as an HDAC inhibitor. In certainembodiments, an anticancer agent is selected from the group consistingof chemotherapeutics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG,Abraxane™, Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-transretinoic acid, amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®,Camptosar®, CeeNU®, Clofarabine, Clolar™, Cytoxan®, daunorubicinhydrochloride, DaunoXome®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®,Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®,hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone,Ixempra®, L-asparaginase, Leukeran®, liposomal Ara-C, L-PAM, Lysodren,Matulane®, mithracin, Mitomycin-C, Myleran®, Navelbine®, Neutrexin®,nilotinib, Nipent®, Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®,Paraplatin®, Platinol®, prolifeprospan 20 with carmustine implant,Sandostatin®, Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA,Trisenox®, Valstar®, Velban®, Vidaza™, vincristine sulfate, VM 26,Xeloda® and Zanosar®); biologics (such as Alpha Interferon, BacillusCalmette-Guerin, Bexxar®, Campath®, Ergamisol®, Erlotinib, Herceptin®,Interleukin-2, Iressa®, lenalidomide, Mylotarg®, Ontak®, Pegasys®,Revlimid®, Rituxan®, Tarceva™ Thalomid®, Tykerb®, Velcade® andZevalin™); corticosteroids, (such as dexamethasone sodium phosphate,DeltaSone® and Delta-Cortef®); hormonal therapies (such as Arimidex®,Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®,Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, Plenaxis™ andZoladex®); and radiopharmaceuticals (such as Iodotope®, Metastron®,Phosphocol® and Samarium SM-153).

In another embodiment, the other therapeutic agent is a chemotherapeuticagent (also referred to as an anti-neoplastic agent oranti-proliferative agent), selected from the group including analkylating agent; an antibiotic; an anti-metabolite; a detoxifyingagent; an interferon; a polyclonal or monoclonal antibody; an EGFRinhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone;a mitotic inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitors; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase or a protein methyltransferase), a cytidine analoguedrug or any chemotherapeutic, anti-neoplastic or anti-proliferativeagent listed in www.cancer.org/docroot/cdg/cdg_0.asp.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary antibiotics include, but are not limited to, doxorubicin(Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone);bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal(DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin(Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin(Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodinel 31 tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Histone Deacetylase Inhibitors include, but are not limited to,vorinostat (Zolinza).

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron);abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus Torisel); rapamune, ridaforolimus; orAP23573.

Exemplary multi-kinase inhibitors include, but are not limited to,sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474;PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol;seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412;bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244);SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristine, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 Mpotassium oxonate), or lovastatin.

In another aspect, the other therapeutic agent is a chemotherapeuticagent or a cytokine such as G-CSF (granulocyte colony stimulatingfactor).

In yet another aspect, the other therapeutic agents can be standardchemotherapy combinations such as, but not restricted to, CMF(cyclophosphamide, methotrexate and 5-fluorouracil), CAF(cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin andcyclophosphamide), FEC (5-fluorouracil, epirubicin, andcyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, andpaclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP),Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molarratio of 1:0.4:1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar™),CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone orprednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin,oncovin, prednisone or prednisolone), or CMFP (cyclophosphamide,methotrexate, 5-fluorouracil and prednisone).

In another aspect, the other therapeutic agents can be an inhibitor ofan enzyme, such as a receptor or non-receptor kinase. Receptor andnon-receptor kinases are, for example, tyrosine kinases orserine/threonine kinases. Kinase inhibitors described herein are smallmolecules, polynucleic acids, polypeptides, or antibodies.

Exemplary kinase inhibitors include, but are not limited to, Bevacizumab(targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux(targets Erb1), Imatinib/Gleevic (targets Bcr-Abl), Trastuzumab (targetsErb2), Gefitinib/Iressa (targets EGFR), Ranibizumab (targets VEGF),Pegaptanib (targets VEGF), Erlotinib/Tarceva (targets Erb1), Nilotinib(targets Bcr-Abl), Lapatinib (targets Erb1 and Erb2/Her2),GW-572016/lapatinib ditosylate (targets HER2/Erb2), Panitumumab/Vectibix(targets EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targetsincluding RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166(targets EGFR), Canertinib/CI-1033 (targets EGFR),Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200(targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR),PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targetsVEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targetsFLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targetsSRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targetsJAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targetsJAK), sorafenib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-B, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src),AC-220 (targets Flt3), AC-480 (targets all HER proteins, “panHER”),Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), Denosumab(targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534(multiple targets including Flt3).

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, Rapamune (targets mTOR/FRAP1), Deforolimus (targets mTOR),Certican/Everolimus (targets mTOR/FRAP1), AP23573 (targets mTOR/FRAP1),Eril/Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK),Seliciclib/CYC202/Roscovitrine (targets CDK), SNS-032/BMS-387032(targets CDK), Ruboxistaurin (targets PKC), Pkc412 (targets PKC),Bryostatin (targets PKC), KAI-9803 (targets PKC), SF1126 (targets PI3K),VX-680 (targets Aurora kinase), Azd1152 (targets Aurora kinase),Arry-142886/AZD-6244 (targets MAP/MEK), SCIO-469 (targets MAP/MEK),GW681323 (targets MAP/MEK), CC-401 (targets JNK), CEP-1347 (targetsJNK), and PD 332991 (targets CDK). Other examples of the othertherapeutic agents and combination therapy can be found in, e.g.,co-owned US Provisional Application No. 61/61/785,446 with the title“Combination Therapy For Treating Cancer” filed Mar. 14, 2013 (AttorneyDocket No. 41478-520P01US), the contents of which are herebyincorporated by reference in its entirety.

As used herein, “combination therapy” or “co-therapy” includes theadministration of a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, and at least a second agent as part of a specifictreatment regimen intended to provide the beneficial effect from theco-action of these therapeutic agents. The beneficial effect of thecombination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may be, but generally is not, intended toencompass the administration of two or more of these therapeutic agentsas part of separate monotherapy regimens that incidentally andarbitrarily result in the combinations of the present invention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, wherein each therapeuticagent is administered at a different time, as well as administration ofthese therapeutic agents, or at least two of the therapeutic agentsconcurrently, or in a substantially simultaneous manner. Simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical. Therapeutic agents may also be administered inalternation.

The combination therapies featured in the present invention can resultin a synergistic effect in the treatment of a disease or cancer. A“synergistic effect” is defined as where the efficacy of a combinationof therapeutic agents is greater than the sum of the effects of any ofthe agents given alone. A synergistic effect may also be an effect thatcannot be achieved by administration of any of the compounds or othertherapeutic agents as single agents. The synergistic effect may include,but is not limited to, an effect of treating cancer by reducing tumorsize, inhibiting tumor growth, or increasing survival of the subject.The synergistic effect may also include reducing cancer cell viability,inducing cancer cell death, and inhibiting or delaying cancer cellgrowth.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

In another aspect, the compounds described herein, pharmaceuticallyacceptable salts thereof, and/or the compositions described herein maybe administered in combination with radiation therapy. Radiation therapycan also be administered in combination with a composition of thepresent invention and another chemotherapeutic agent described herein aspart of a multiple agent therapy.

The compounds of the instant invention can also be utilized to treat orprevent neurologic diseases or disorders in monotherapy or combinationtherapy. Neurologic diseases or disorders that may be treated with thecompounds of this invention include epilepsy, schizophrenia, bipolardisorder or other psychological and/or psychiatric disorders,neuropathies, skeletal muscle atrophy, and neurodegenerative diseases,e.g., a neurodegenerative disease. Exemplary neurodegenerative diseasesinclude: Alzheimer's, Amyotrophic Lateral Sclerosis (ALS), andParkinson's disease. Another class of neurodegenerative diseasesincludes diseases caused at least in part by aggregation ofpoly-glutamine. Diseases of this class include: Huntington's Diseases,Spinalbulbar Muscular Atrophy (SBMA or Kennedy's Disease)Dentatorubropallidoluysian Atrophy (DRPLA), Spinocerebellar Ataxia 1(SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease (MJD;SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7 (SCA7),and Spinocerebellar Ataxia 12 (SCA12).

Any other disease in which epigenetic methylation, which is mediated byDOT1, plays a role may be treatable or preventable using compounds andmethods described herein.

In one aspect, the compound suitable for the method of the invention,e.g., a DOT1L inhibitor, is a compound of Formula (I) or (II) describedherein, or a pharmaceutically acceptable salt or ester thereof:

The invention also relates to a pharmaceutical composition of atherapeutically effective amount of a compound of any of the Formulaedisclosed herein and a pharmaceutically acceptable carrier.

The invention also relates to a pharmaceutical composition of atherapeutically effective amount of a salt of a compound of any of theFormulae disclosed herein and a pharmaceutically acceptable carrier.

The invention also relates to a pharmaceutical composition of atherapeutically effective amount of a hydrate of a compound of any ofthe Formulae disclosed herein and a pharmaceutically acceptable carrier.

The invention also relates to a pharmaceutical composition of atherapeutically effective amount of a compound selected from compounds1-89, 101-104, and 107-114 and pharmaceutically acceptable salts thereofand a pharmaceutically acceptable carrier. The invention also relates toa pharmaceutical composition of a therapeutically effective amount of asalt of a compound selected from compounds 1-89, 101-104, and 107-114and pharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable carrier. The invention also relates to a pharmaceuticalcomposition of a therapeutically effective amount of a hydrate of acompound selected from compounds 1-89, 101-104, and 107-114 andpharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable carrier.

The present invention provides methods of treating or preventing cancer.The present invention provides methods of treating cancer. The presentinvention also provides methods of preventing cancer. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of the compound of any of the Formulae disclosedherein. The cancer can be a hematological cancer. Preferably, the canceris leukemia. More preferably, the cancer is acute myeloid leukemia,acute lymphocytic leukemia or mixed lineage leukemia.

The present invention provides methods of treating or preventing adisease or disorder mediated by translocation of a gene on chromosome 11q23. The present invention provides methods of treating a disease ordisorder mediated by translocation of a gene on chromosome 11q23. Thepresent invention also provides methods of preventing a disease ordisorder mediated by translocation of a gene on chromosome 11 q23. Themethod includes administering to a subject in need thereof atherapeutically effective amount of the compound of any of the Formulaedisclosed herein.

The present invention provides methods of treating or preventing adisease or disorder in which DOT1L-mediated protein methylation plays apart or a disease or disorder mediated by DOT1L-mediated proteinmethylation. The present invention provides methods of treating adisease or disorder in which DOT1L-mediated protein methylation plays apart or a disease or disorder mediated by DOT1L-mediated proteinmethylation. The present invention also provides methods of preventing adisease or disorder in which DOT1L-mediated protein methylation plays apart or a disease or disorder mediated by DOT1L-mediated proteinmethylation. The method includes administering to a subject in needthereof a therapeutically effective amount of the compound of any of theFormulae disclosed herein.

The present invention provides methods of inhibiting DOT1L activity in acell. The method includes contacting the cell with an effective amountof one or more of the compound of any of the Formulae disclosed herein.

Still another aspect of the invention relates to a method of reducingthe level of Histone H3 Lysine residue 79 (H3-K79) methylation in acell. The method includes contacting a cell with a compound of thepresent invention. Such method can be used to ameliorate any conditionwhich is caused by or potentiated by the activity of DOT1L throughH3-K79 methylation.

The present invention relates to use of the compounds disclosed hereinin preparation of a medicament for treating or preventing cancer. Theuse includes a compound of any of the Formulae disclosed herein foradministration to a subject in need thereof in a therapeuticallyeffective amount. The cancer can be a hematological cancer. Preferably,the cancer is leukemia. More preferably, the cancer is acute myeloidleukemia, acute lymphocytic leukemia or mixed lineage leukemia.

The present invention provides use of the compounds disclosed herein inpreparation of a medicament for treating or preventing a disease ordisorder mediated by translocation of a gene on chromosome 11q23. Theuse includes a compound of any of the Formulae disclosed herein foradministration to a subject in need thereof in a therapeuticallyeffective amount.

The present invention provides use of the compounds disclosed herein inpreparation of a medicament for treating or preventing a disease ordisorder in which DOT1L-mediated protein methylation plays a part or adisease or disorder mediated by DOT1L-mediated protein methylation. Theuse includes a compound of any of the Formulae disclosed herein foradministration to a subject in need thereof in a therapeuticallyeffective amount.

The present invention provides use of the compounds disclosed herein forinhibiting DOT1L activity in a cell. The use includes contacting thecell with an effective amount of one or more of the compound of any ofthe Formulae disclosed herein.

Still another aspect of the invention relates to a use of the compoundsdisclosed herein for reducing the level of Histone H3 Lysine residue 79(H3-K79) methylation in a cell. The use includes contacting a cell witha compound of the present invention. Such use can ameliorate anycondition which is caused by or potentiated by the activity of DOT1Lthrough H3-K79 methylation.

In the formulae presented herein, the variables can be selected from therespective groups of chemical moieties later defined in the detaileddescription.

In addition, the invention provides methods of synthesizing theforegoing compounds. Following synthesis, a therapeutically effectiveamount of one or more of the compounds can be formulated with apharmaceutically acceptable carrier for administration to a mammal,particularly humans, for use in modulating an epigenetic enzyme. Incertain embodiments, the compounds of the present invention are usefulfor treating, preventing, or reducing the risk of cancer or for themanufacture of a medicament for treating, preventing, or reducing therisk of cancer. Accordingly, the compounds or the formulations can beadministered, for example, via oral, parenteral, otic, ophthalmic,nasal, or topical routes, to provide an effective amount of the compoundto the mammal.

Representative compounds of the present invention include compoundslisted in Tables 1 and 2.

TABLE 1 Com- pound no. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

TABLE 2 Com- Nominal Approx. pound mass Retention No. Structure (Da)Time (min) 101

320 10.4 102

304 13.7 103

288 18.5 104

286 19.6 105

578 20   106

520 22.5 107

578 24.5 108

578 25   A

562 26   109

329 28.5 110

536 18.3 111

592 12.8 13.2 (split peak) 112

550 11.3 113

313 34.3 114

447 28.7

As used herein, the term “isolated” or the expression “in an isolatedform” means substantially separated from other components with which thecompound may be found as it occurs in nature. A compound can be isolatedwithout necessarily being purified. In one embodiment, the isolatedcompound of Formula (I) or (II) is a synthesized compound. In anotherembodiment, the compound of Formula (I) or (II) is a metabolite and isisolated from other components with which the compound may be found asit occurs in a natural environment, e.g., cells of a mammal, e.g., ahuman or appropriate non-human mammal, such as primate, mouse, rat, dog,cat, cow, horse, goat, and camel. In embodiments, the compound or saltof the present invention has a purity of not less than 40%, not lessthan 50%, not less than 55%, not less than 60%, not less than 65%, notless than 70%, not less than 75%, not less than 80%, not less than 85%,not less than 90%, not less than 92%, not less than 95%, not less than97%, or not less than 99%.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. For example, in Formula (I), each hydroxyl of the moiety —(OH)₁can be bonded to any ring atom of the benzimidazole ring (such as acarbon in the benzene ring or a nitrogen atom in the imidazole ring), aslong as valence permits and/or the compound thus formed is stable. Whena substituent is listed without indicating the atom via which suchsubstituent is bonded to the rest of the compound of a given formula,then such substituent may be bonded via any atom in such formula.Combinations of substituents and/or variables are permissible, but onlyif such combinations result in stable compounds.

When any variable occurs more than one time in any constituent orformula for a compound, its definition at each occurrence is independentof its definition at every other occurrence. Thus, for example, if agroup is shown to be substituted with 0-2 R moieties, then the group mayoptionally be substituted with up to two R moieties and R at eachoccurrence is selected independently from the definition of R. Also,combinations of substituents and/or variables are permissible, but onlyif such combinations result in stable compounds.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

Compounds of the present invention that contain nitrogens includeN-oxides (which can be designated as N→O or N⁺—O⁻). The N-oxides of thepresent invention can be generated by, e.g., treating compounds containgnitrogens, e.g., Compound A, with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides).Furthermore, in other instances, the compounds of the present inventioncan be N-hydroxy (i.e., N—OH) compounds. For example, N-hydroxycompounds can be prepared by oxidation of the parent amine, e.g.,Compound A, by an oxidizing agent such as mCPBA.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present invention maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present invention, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), amine-enamine andenamine-enamine. Benzimidazoles also exhibit tautomerism, when thebenzimidazole contains one or more substituents in the 4, 5, 6 or 7positions, the possibility of different isomers arises. For example,2,5-dimethyl-1H-benzo[d]imidazole can exist in equilibrium with itsisomer 2,6-dimethyl-1H-benzo[d]imidazole via tautomerization.

Another example of tautomerism is shown below.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Compounds of the invention may be crystalline, semi-crystalline,non-crystalline, amorphous, mesomorphous, etc.

The compounds of any of the Formulae disclosed herein include thecompounds themselves, as well as their salts, their solvates, and theirprodrugs, if applicable. A salt, for example, can be formed between ananion and a positively charged group (e.g., amino) on the compound orinhibitor (e.g., a substituted nucleoside compound such as a substitutedpurine compound). Suitable Inions include chloride, bromide, iodide,sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate,malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate,lactate, naphthalenesulfonate, and acetate. Likewise, a salt can also beformed between a cation and a negatively charged group (e.g.,carboxylate) on the compound or inhibitor (e.g., a substitutednucleoside compound such as a substituted purine compound). Suitablecations include sodium ion, potassium ion, magnesium ion, calcium ion,and an ammonium cation such as tetramethylammonium ion. The compound orinhibitor (e.g., a substituted nucleoside compound such as a substitutedpurine compound) also include those salts containing quaternary nitrogenatoms. Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing active substituted nucleoside compound such as asubstituted purine compounds of the invention.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include hemihydrates, monohydrates, dihydrates,trihydrates, etc. Nonlimiting examples of solvates include ethanolsolvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O. A hemihydrate isformed by the combination of one molecule of water with more than onemolecule of the substance in which the water retains its molecular stateas H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) or (II) are substituted purine compounds, and have Formula(I) or (II) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

The present invention also provides methods for the synthesis of thecompounds of any of the Formulae disclosed herein. The present inventionalso provides detailed methods for the synthesis of various disclosedcompounds of the present invention according to the schemes and theExamples described in WO2012/075381, WO2012/075492, WO2012/082436, andWO2012/75500, the contents of which are hereby incorporated by referencein their entireties.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester, or prodrug thereof.

Compounds of the present invention can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001;Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999; R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

One of ordinary skill in the art will note that, during the reactionsequences and synthetic schemes described herein, the order of certainsteps may be changed, such as the introduction and removal of protectinggroups.

One of ordinary skill in the art will recognize that certain groups mayrequire protection from the reaction conditions via the use ofprotecting groups. Protecting groups may also be used to differentiatesimilar functional groups in molecules. A list of protecting groups andhow to introduce and remove these groups can be found in Greene, T. W.,Wuts, P. G. M., Protective Groups in Organic Synthesis, 3^(rd) edition,John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

For the hydroxyl moiety: TBS, benzyl, THP, Ac

For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allylester

For amines: Cbz, BOC, DMB

For diols: Ac (×2) TBS (×2), or when taken together acetonides

For thiols: Ac

For benzimidazoles: SEM, benzyl, PMB, DMB

For aldehydes: di-alkyl acetals such as dimethoxy acetal or diethylacetyl.

In the reaction schemes described herein, multiple stereoisomers may beproduced. When no particular stereoisomer is indicated, it is understoodto mean all possible stereoisomers that could be produced from thereaction. A person of ordinary skill in the art will recognize that thereactions can be optimized to give one isomer preferentially, or newschemes may be devised to produce a single isomer. If mixtures areproduced, techniques such as preparative thin layer chromatography,preparative HPLC, preparative chiral HPLC, or preparative SFC may beused to separate the isomers.

The following abbreviations are used throughout the specification andare defined below:

-   -   AA ammonium acetate    -   Ac acetyl    -   ACN acetonitrile    -   AcOH acetic acid    -   atm atmosphere    -   Bn benzyl    -   BOC tert-butoxy carbonyl    -   BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium        hexafluorophosphate    -   Cbz benzyloxycarbonyl    -   COMU        (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium        hexafluorophosphate    -   d days    -   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCE 1,2 dichloroethane    -   DCM dichloromethane    -   DEA diethylamine    -   DEAD diethyl azodicarboxylate    -   DIAD diisopropyl azodicarboxylate    -   DiBAL-H diisobutylalumininium hydride    -   DIPEA N,N-diisopropylethylamine (Hunig's base)    -   DMAP N,N-dimethyl-4-aminopyridine    -   DMB 2,4 dimethoxybenzyl    -   DMF dimethylformamide    -   DMSO dimethylsulfoxide    -   DPPA diphenylphosphoryl azide    -   EA or EtOAc ethylacetate    -   EDC or EDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide    -   ELS Evaporative Light Scattering    -   ESI− Electrospray negative mode    -   ESI+ Electrospray positive mode    -   Et₂O diethyl ether    -   Et₃N or TEA triethylamine    -   EtOH ethanol    -   FA formic acid    -   FC flash chromatography    -   h hours    -   H₂O water    -   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HCl hydrochloric acid    -   HOAT 1-hydroxy-7-azabenzotriazole    -   HOBt 1-hydroxybenzotriazole    -   HOSu N-hydroxysuccinimide    -   HPLC high performance liquid chromatography    -   Inj. Vol. injection volume    -   I.V. or IV intravenous    -   KHMDs potassium hexamethyldisilazide    -   LC/MS or LC-MS liquid chromatography mass spectrum    -   LDA lithium diisopropylamide    -   LG leaving group    -   LiHMs lithium hexamethyldisilazide    -   M Molar    -   m/z mass/charge ratio    -   m-CPBA meta-chloroperbenzoic acid    -   MeCN acetonitrile    -   MeOD d₄-methanol    -   MeOH methanol    -   MgSO₄ magnesium sulfate    -   min minutes    -   MS mass spectrometry or mass spectrum    -   Ms mesyl    -   MsCl methanesulfonyl chloride    -   MsO mesylate    -   MWI microwave irradiation    -   Na₂CO₃ sodium carbonate    -   NaHCO₃ sodium bicarbonate    -   NaHMDs sodium hexamethyldisilazide    -   NaOH sodium hydroxide    -   NIS N-iodosuccinimide    -   NBS N-bromosuccinimide    -   NCS N-chlorosuccinimide    -   NMR Nuclear Magnetic Resonance    -   o/n or O/N overnight    -   PE petroleum ether    -   PG protecting group    -   PKMT protein lysine methyltransferase    -   PMB para-methoxybenzyl    -   PMT protein methyltransferase    -   PPAA 1-propanephosphonic acid cyclic anhydride    -   ppm parts per million    -   prep HPLC preparative high performance liquid chromatography    -   prep TLC preparative thin layer chromatography    -   p-TsOH para-toluenesulfonic acid    -   rt or RT room temperature    -   SAH S-adenosylhomocysteine    -   SAM S-adenosylmethionine    -   SAR structure activity relationship    -   Selectfluor®        1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane        bis(tetrafluoroborate),        N-Chloromethyl-N′-fluorotriethylenediammonium        bis(tetrafluoroborate)    -   SEM 2-(trimethylsilyl)ethoxymethyl    -   SEMCl (trimethylsilyl)ethoxymethyl chloride    -   SFC supercritical chromatography    -   SGC silica gel chromatography    -   SPR surface plasmon resonance    -   STAB sodium triacetoxyborohydride    -   TBAF tetra-n-butylammonium fluoride    -   TFA trifluoroacetic acid    -   TfO triflate    -   THF tetrahydrofuran    -   THP tetrahydropyran    -   TLC thin layer chromatography    -   Ts tosyl    -   TsOH tosic acid    -   UV ultraviolet

The invention provides methods for making the compounds of theinvention. The following schemes depict exemplary chemistries availablefor synthesizing the compounds of the invention.

5′-Amino purine-ribose intermediates (A-V) can be synthesized asdepicted in Scheme 1 above. A suitable protected 6-Cl adenosinederivative (A-I) is converted into a 6-amino derivative (A-II) bytreatment with the appropriate amine (including ammonia) in the presenceof a base such as Et₃N, K₂CO₃ or Hunig's base in solvent such as MeCN orDMF, THF, iPrOH or a mixture thereof. If required, the reaction may beheated to 100° C. (if the temperature required is greater than theboiling point of one or more of the components in the mixture, thereaction may be performed in a sealed tube). The R groups in the schememay represent alkyl protecting groups (e.g., 2, 4 dimethoxybenzyl). The6-amino product (A-II) may be transformed into the 5′-azido intermediate(A-III) by converting the 5′-hydroxyl group into a leaving group such asMsO (i.e., CH₃S(O)₂O) by treatment with methanesulfonyl chloride (MsCl)in the presence of a base such as Et₃N, pyridine or K₂CO₃ in an inertsolvent such as CH₂Cl₂, THF, MeCN, DMF or a mixture thereof. The5′-leaving group is then displaced with azide anion from NaN₃ in aninert solvent such as DMF. Alternatively (A-II) may be directlytransformed into (A-III) by treatment with DPPA, Ph₃P, and DIAD in asolvent such as THF. The azido group of (A-III) may be reduced to theprimary amine (A-IV) by reduction with H₂ in the presence of a metalcatalyst (e.g. Pd/C, PtO₂) or by a Staudinger reaction with a phosphinesuch as Ph₃P or PMe₃. The primary amine (A-IV) may be converted into thesecondary amine (A-V) by treatment with the appropriate ketone oraldehyde in the presence of a suitable reducing agent such as NaBH(OAc)₃or NaCNBH₃. Additional reagents such as Ti(OiPr)4 may be added.

Alternatively the 5′-hydroxy intermediate (A-II) may be treated with thesulfonamide (A-VI), DEAD and Ph₃P in an inert solvent such as THF. Theresultant sulfonamide product may then be treated with benzenethiol inthe presence of a base such as K₂CO₃, Cs₂CO₃ to give the secondary amine(A-V).

These reaction sequences above may also be applied to lyxose derivativesstarting from (A-VII) to give the diastereomer with oppositeconfiguration at the 5′ position.

A similar set of reaction sequences may be employed for 2′-deoxy, or3′-deoxy, or substituted ribose or lyxose (A-VIII) above to obtain5′-amino purine-ribose/lyxose intermediates.

An alternative method for introduction of a 6-NH₂ group, as shown below,is via treating (A-IX) derivatives with NaN₃ to produce a 6-azidointermediate followed by reduction to the NH₂ moiety (A-X) with atrialkyl phosphine such as PMe₃ or PPh₃.

Cyclobutanes of formulae (G-VIII), (G-XV) and (G-XXI) may be synthesizedas depicted in Scheme 2. The alkenyl esters (G-I) may be subjected to a[2+2] cycloaddition with trichloroacetyl chloride in the presence ofZn/Cu couple in an inert solvent such as Et₂O, DME, THF or a mixturethereof. Alternatively the [2+2] cycloaddition reaction may be performedusing Zn dust under sonication conditions. The dichlorides (G-II) arereduced via treatment with Zn powder in the presence of a proton donorsuch as NH₄Cl in a solvent such as MeOH. The cyclobutanones (G-IV)(which include (G-III)) may be further elaborated by treatment with aphosphonate (G-V) to give the α, β unsaturated esters (G-VI). The acid(VI) is converted to the Weinreb amide (G-VII) under standard conditions(e.g. iso-butyl chloroformate, Hunig's base, N,O-dimethylhydroxylamine). The double bond may then be reduced via hydrogenationusing H₂ in the presence of a metal catalyst such as Pd/C, PtO₂ orPd(OH)₂ to give the cyclobutane intermediates (G-VIII).

The cyclobutanones (G-IX) may be treated with the Wittig reagent (G-X)to give the cyclobutane enol ether (G-XI) which upon deprotection givesthe corresponding acid (G-XII).

The cyclobutanones (G-IX) may also be treated with the stabilizedphosphonate (G-V) in the presence of a base such as KOtBu, LDA, NaHMDS,KHMDS or LiHMDS or with Et₃N in the presence of LiCl in an inert solventto give the α, β unsaturated ester (G-XIII) which can be reduced to the(G-XIV) by treatment with H₂ in the presence of a metal catalyst such asPd/C, Pd(OH)₂ or PtO₂ in an inert solvent. The acid functionality of(G-XIV) may be converted into the corresponding Weinreb amide bytreatment with N,O-dimethylhydroxylamine in presence of a suitablecoupling agent such as iso-butylchloroformate and a base such as Hunig'sbase to give (G-XV).

The cyclobutanones (G-XVI) may also be treated withN,O-dimethylhydroxylamine in presence of a suitable coupling agent suchas iso-butylchloroformate and a base such as Hunig's base to give thecorresponding Weinreb amide (G-XVII) which upon reductive amination withan ammonia equivalent followed by deprotection as needed gives the amine(G-XVIII). Suitable Immonia equivalents include benzhydryl amine, NH₃,NH₄Cl, BnNH₂, PMB-NH₂, 2,4 DMB-NH₂ which may be treated with the ketone(G-XVII) and a suitable reducing agent such as NaCN(BH₃) or Na(OAc)₃BHin the presence of an acid if required such as HCl or AcOH. Protectinggroups on the reductive amination products may be removed by methodsknown to those of ordinary skill in the art. Alternatively the ketone(G-XVII) can be treated with hydroxyl amine to form the correspondingoxime which then can be reduced with H₂ in the presence of a metalcatalyst such as Pd/C, PtO₂ or Pd(OH)₂ to give the intermediate(G-XVIII).

The cyclobutane (G-IV) may converted into the amine (G-XXI) via amulti-step sequence involving treating (G-IV) with the phosphorane (G-V)to produce the enol ether (G-XIX). Treatment of (G-XIX) with which isthen N,O-dimethylhydroxylamine in presence of a suitable coupling agentsuch as iso-butyl chloroformate and a base such as Hunig's base to givethe corresponding Weinreb amide (G-XX) which after aqueous hydrolysis ofthe enol ether (e.g. TsOH/H₂O, HCl/H₂O) and reductive amination with anammonia equivalent followed by deprotection as needed gives the amine(G-XXI). Suitable Immonia equivalents include benzhydryl amine, NH₃,NH₄Cl, BnNH₂, PMB-NH₂, 2,4 DMB-NH₂. Suitable reducing agents for thereductive amination include NaCN(BH₃) or Na(OAc)₃BH used in the presenceof an acid if required such as HCl or AcOH. Protecting groups on thereductive amination products may be removed by methods known to those ofordinary skill in the art.

The formula (K-V)

(K-V) in Scheme 3 above represents the intermediates (K-I through K-IV)below and their corresponding 2′- or 3′-deoxy intermediates, whosesyntheses are described in, e.g., WO 2012/075381.

As shown in Scheme 3, the ketones (K-VI), (K-VII) and (K-VIII) and thealdehydes (K-XII), (K-XIII) and (K-XIV) are converted into thecorresponding benzimidazoles (K-IX) and (K-XV), ureas (K-X) and (K-XVI)and amides (K-XI) and (K-XVII) via reductive amination with (K-V). Thereductive amination can be performed with a suitable reducing agent suchas NaCN(BH₃) or Na(OAc)₃BH in the presence of an acid if required suchas HCl or AcOH or a Lewis acid/dehydrating agent such as Ti(OiPr)₄ orMgSO₄.

In one embodiment, Compound A may be synthesized by routes depicted inscheme 1A below. The process is a 4-step synthesis including apurification step to produce pure Compound A:(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof. More detailed synthetic disclosure can be foundin, e.g., co-owned U.S. application Ser. No. 14/210,888, filed Mar. 14,2014 (Attorney Docket No.: EPIZ-023/001US), the content of which ishereby incorporated by reference in its entirety. The variables in thechemical formula included in Scheme 1A (e.g., R₁, R₂, l′, m′, n′, p′,q′, r′, t′, u′, v′, and w′ are as defined herein for Formula II).

Compounds of the invention, such as those selected from compounds 1-89,101-104, and 107-114 and pharmaceutically acceptable salts thereof, canbe synthesized via methods similar to those for Compound A. In someembodiments, compounds of the invention, such as N-oxides or N-hydroxycompounds of Compound A can be synthesized by oxidizing Compound A withan oxidizing agent such as mCPBA, for example, step 5 of Scheme 1A aboveis an oxidization step. In some embodiments, a compound of theinvention, such as a monohydroxylated-Compound A, is produced viaenzymatic reactions either in vivo or in vitro, for example, step 5 ofScheme 1A above is an in vivo or in vitro metabolization step. In otherembodiments, a hydroxylated-Compound A, is synthesized via a multi-stepreaction, e.g., step 5 of Scheme 1A above includes multiple steps withsuitable reaction conditions to afford the hydroxylated compound ofinterest. In some embodiments, a compound of the invention, such asCompound No. 111, is produced via oxidation, for example, step 5 ofScheme 1A above includes either chemical or enzymatic reactions orcombination thereof to oxidize Compound A to a corresponding carboxylicacid. In some embodiments, a compound of the invention, such as CompoundNo. 110 or 112, is produced via dealkylation and hydroxylation oroxidation, for example, step 5 of Scheme 1A above includes eitherchemical or enzymatic reactions or combination thereof to (i) afford thecorresponding dealkylated Compound A and then the correspondinghydroxylated compound or carboxylic acid, or (ii) to afford thecorresponding hydroxylated compound or carboxylic acid and then thecorresponding dealkylated compound.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components, and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions can be conducted simultaneously.

Compounds suitable for the methods of the invention, once produced, canbe characterized using a variety of assays known to those skilled in theart to determine whether the compounds have biological activity. Forexample, the molecules can be characterized by conventional assays,including but not limited to those assays described below, to determinewhether they have a predicted activity, binding activity and/or bindingspecificity.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen themolecules described herein for activity, using techniques known in theart. General methodologies for performing high-throughput screening aredescribed, for example, in Devlin (1998) High Throughput Screening,Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays canuse one or more different assay techniques including, but not limitedto, those described herein.

To further assess a compound's drug-like properties, measurements ofinhibition of cytochrome P450 enzymes and phase II metabolizing enzymeactivity can also be measured either using recombinant human enzymesystems or more complex systems like human liver microsomes. Further,compounds can be assessed as substrates of these metabolic enzymeactivities as well. These activities are useful in determining thepotential of a compound to cause drug-drug interactions or generatemetabolites that retain or have no useful antimicrobial activity.

To get an estimate of the potential of the compound to be orallybioavailable, one can also perform solubility and Caco-2 assays. Thelatter is a cell line from human epithelium that allows measurement ofdrug uptake and passage through a Caco-2 cell monolayer often growingwithin wells of a 24-well microtiter plate equipped with a 1 micronmembrane. Free drug concentrations can be measured on the basolateralside of the monolayer, assessing the amount of drug that can passthrough the intestinal monolayer. Appropriate controls to ensuremonolayer integrity and tightness of gap junctions are needed. Usingthis same system one can get an estimate of P-glycoprotein mediatedefflux. P-glycoprotein is a pump that localizes to the apical membraneof cells, forming polarized monolayers. This pump can abrogate theactive or passive uptake across the Caco-2 cell membrane, resulting inless drug passing through the intestinal epithelial layer. These resultsare often done in conjunction with solubility measurements and both ofthese factors are known to contribute to oral bioavailability inmammals. Measurements of oral bioavailability in animals and ultimatelyin man using traditional pharmacokinetic experiments will determine theabsolute oral bioavailability.

Experimental results can also be used to build models that help predictphysical-chemical parameters that contribute to drug-like properties.When such a model is verified, experimental methodology can be reduced,with increased reliance on the model predictability.

The present invention also provides pharmaceutical compositionscomprising a compound of any of the Formulae disclosed herein incombination with at least one pharmaceutically acceptable excipient orcarrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not as high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic selected for administration.Therapeutically effective amounts for a given situation can bedetermined by routine experimentation that is within the skill andjudgment of the clinician. In a preferred aspect, the disease orcondition to be treated is cancer. In another aspect, the disease orcondition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug interaction(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present invention can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g, a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., acetate, propionate or other ester.

The compounds of the present invention can also be prepared as prodrugs,for example, pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs in thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy or carbonyl group is bonded to any group that may becleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, N.Y.-Oxford (1985).

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

In the synthetic schemes described herein, compounds may be drawn withone particular configuration for simplicity. Such particularconfigurations are not to be construed as limiting the invention to oneor another isomer, tautomer, regioisomer or stereoisomer, nor does itexclude mixtures of isomers, tautomers, regioisomers or stereoisomers.

Compounds described herein are assayed for modulation of activity, forexample, histone methylation, modulation of cell growth and/or IC₅₀,described in the examples below.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

Example 1: Synthesis and/or Isolation of Compounds

Compound A was synthesized by the methods described in WO 2012/075381 orco-owned U.S. Provisional Application Ser. No. 61/799,147, filed Mar.15, 2013 (Attorney Docket No.: 41478-523P01US), the contents of whichare hereby incorporated by reference in their entireties.

Compounds of the invention, such as those selected from compounds 1-89,101-104, and 107-114 and pharmaceutically acceptable salts thereof, canbe synthesized via methods similar to those for Compound A. Certaincompounds in Table 1 or 2, such as N-oxides of Compound A can besynthesized by oxidizing Compound A with an oxidizing agent such asmCPBA.

Compounds of the invention, such as metabolites of Compound A, can alsobe generated by separating these compounds from their naturallyoccurring environment, by e.g., chromatography, methods describedherein, or other known purification methods.

Example 2: Preclinical Pharmacokinetic Evaluation of Compound A

Compound A:((2R,3R,4S,5R)-2-(6-(amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol)is a DOT1L histone methyltransferase inhibitor currently in developmentfor the treatment of MLL-rearranged leukemias. Described below are thepreclinical pharmacokinetics and metabolism studies conducted forCompound A, an aminonucleoside analog with exquisite target potency andselectivity that has shown robust and durable tumor growth inhibition inpreclinical models. The in vivo pharmacokinetics in mouse, rat and dogwere characterized and applied to the prediction of human PK parametersand exposure, using various interspecies scaling approaches. Preclinicalpharmacokinetics indicates Compound A has moderate CL with asteady-state volume of distribution 2-3 fold higher than total bodywater. Corresponding in vitro ADME parameters were also studied andutilized for in vitro-in vivo extrapolation purposes. There wasreasonable congruence between microsomal CL and in vivo CL implicatinghepatic oxidative metabolism as the predominant elimination route inpreclinical species. The metabolic pathways across species were studiedin liver microsomes in which Compound A was metabolized to threemonohydroxylated metabolites (Compounds M1, M3 and M5), oneN-dealkylated product (Compound M4) as well as an N-oxide (Compound M6).None of the metabolites detected in this milieu were unique to human.This first-in-class DOT1L inhibitor with promising therapeutic potentialin MLL-rearranged leukemias is currently in Phase 1 clinical trials.

Compound A inhibits DOT1L with a Ki of 80 pM and displays 37,000-foldselectivity over a panel of other HMTs. Potency was further exemplifiedby treatment in a rat xenograft model of MLL-rearranged leukemia withCompound A, in which continuous intravenous (IV) infusion of Compound Acaused complete tumor regressions that were sustained beyond thecompound infusion period with no significant weight loss or signs oftoxicity (Daigle et al., Potent Inhibition of DOT1L as Treatment forMLL-Fusion Leukemia. Blood 2013; 122(6): 1017-1025).

Understanding of the pharmacokinetic properties along with theremarkable potency of Compound A both in vitro and in vivo promoted thedevelopment of this molecule for acute leukemias bearingMLL-rearrangements. Compound A is currently in Phase I evaluation andrepresents not only the first reported histone methyltransferaseinhibitor to enter human clinical trials, but a further step inunderstanding the link between epigenetic processes and thepathophysiology of cancer.

Methods Chemicals and Reagents

Compound A was synthesized by Epizyme (Daigle et al., Blood 2013;122(6): 1017-1025). All other reagents were purchased from sources asdescribed below.

In Vivo Pharmacokinetics

All animal studies were conducted in accordance with local IACUCstandards.

Pharmacokinetic Study in Mouse.

Pharmacokinetics of Compound A was evaluated in male CD1-mice (28-29grams, male, n=21, purchased from BK Laboratory Animal Co. LTD)following IV bolus administration of doses of 5 mg/kg and oraladministration at doses of 20 mg/kg. Oral and IV doses were administeredby oral gavage, or by tail vein injection in a 10% ethanol and 90%saline vehicle, respectively. For PO dosing, samples were taken at0.167, 0.5, 1, 2, 4, 6 and 24 h p.d. For IV administration, samples weretaken at 0.05, 0.167, 0.5, 1, 2, 4, 6 and 24 h p.d. Blood samples werecentrifuged at 4° C. (2000 g, 5 minutes) to obtain plasma within 15minutes after sample collection. The levels of Compound A werequantitated in mouse plasma by LC-MS/MS analysis.

Pharmacokinetic Study in Rat.

Pharmacokinetics of Compound A was evaluated in Sprague-Dawley rats(male, n=3 for IV bolus and PO studies and n=6 for IV infusion study),purchased from either SLAC Laboratory Animal Co. LTD (IV bolus), VitalRiver Laboratory Animal Co. LTD (PO study), or Charles River CanadaInc., St. Constant, QC, Canada (IV infusion). In the IV bolus study, 1mg/kg doses prepared in 0.4% HPBCD in saline were administered via footdorsal vein injection. At each timepoint, 150 μL of blood was collectedvia tail vein into EDTA-K₂ tubes. The blood samples were maintained inwet ice initially and centrifuged to obtain plasma (2000 g, 4° C., 5minutes) within 15 minutes post sampling. In the PO study, dosesprepared in 10% Ethanol:5% Solutol HS15:85% (5% of dextrose in water)were administered by oral gavage. Samples were collected as describedabove. In the IV infusion study, doses were prepared in 10% PEG400 in0.9% saline and administered into the femoral vein, at a dose of 4.7mg/kg/day for 7 days. 500 μL of blood was collected by jugularvenipuncture at timepoints over 7 days and placed on crushed wet iceuntil centrifugation, which was carried out as soon as practical. Thesamples were centrifuged for 10 minutes at 4° C. at 2700 rpm. Theresultant plasma was separated and frozen immediately over dry ice forLC-MS/MS analysis.

Pharmacokinetic Study in Dog.

Intravenous (IV) pharmacokinetics of Compound A was evaluated in beagledogs (male, n=3, purchased from Beijing Marshall Biotechnology Co. Ltd.)following a single IV administration at a dose of 1 mg/kg. IV doses wereadministered by a single intravenous infusion over 1 minute into thecephalic vein in a 10% ethanol and 90% saline vehicle. At designatedtime points (predose, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h p.d.), theanimals were restrained manually, and approximately 0.5 mL blood pertime point was collected from non-injected cephalic vein into pre-coldEDTA-K₂ tubes. Blood samples were put on wet ice and centrifuged at 4°C. to obtain plasma within 15 minutes of sample collection. The levelsof Compound A were quantitated in dog plasma by LC-MS/MS analysis.

LC-MS/MS Bioanalysis & Pharmacokinetic Data Analysis

Compound A was extracted from K₂EDTA plasma by protein precipitationusing an acetonitrile-containing internal standard (a structural analogof Compound A at a concentration of 5 ng/mL). Typically, samples wereinjected onto an LC-MS/MS system using a Waters BEH Phenyl column. Theaqueous mobile phase was water with 0.1% NH₄OH (A), and the organicmobile phase was acetonitrile with 0.1% NH₄OH (B). The gradient was asfollows: 37% B for the first 0.2 min, increased to 44% B from 0.2 to 0.6min, maintained at 44% B for 0.5 min, and decreased to 37% B within 0.05min. The injection volume was 2 uL, and the total run time was 1.5 minwith a flow rate of 0.6 mL/min. The retention time of Compound A was0.85 min. The ionization was conducted in the positive ion mode usingthe MRM transition [M+H]+m/z 563.5 parent ion to m/z 326.3 daughter ion,incorporating a turbo-ionspray interface. Eight to ten calibrationstandards were prepared in blank plasma of the relevant speciesproviding a typical standard curve concentration range of 5-500 ng/mL.

Pharmacokinetic parameters were calculated by noncompartmental methodsusing WinNonlin (version 5.3; Pharsight, St. Louis, Mo.). Parameters arepresented as mean±S.D.

In Vitro Stability Assays in Liver Microsomes and Hepatocytes

Liver microsomes (final protein concentration 0.5 mg/mL), 0.1 Mphosphate buffer at pH 7.4 and Compound A (final concentration of 3 μM;final DMSO concentration of 0.25%) were pre-incubated at 37° C. prior tothe addition of NADPH (final concentration of 1 mM) to initiate thereaction. The final incubation volume was 50 μL. Control incubationswere included for each species where 0.1 M phosphate buffer pH 7.4 wasadded instead of NADPH (minus NADPH). Two control compounds wereincluded for each species. Compound A and controls were incubated for 0,5, 15, 30 and 45 minutes. The control (minus NADPH) was incubated for 45minutes only. The reactions were stopped by transferring 25 μL ofincubate to 50 μL methanol at the appropriate time points. Theincubation plates were centrifuged at 2,500 rpm for 20 minutes at 4° C.to aid protein precipitation.

Human, Beagle dog, Sprague-Dawley rat, and CD-1 mouse cryopreservedhepatocytes were obtained from XenoTech and stored at −150° C. untiluse. The hepatocytes were thawed and prepared according to the vendor'sinstructions, pooled into Krebs Henseleit buffer (KHB, pH 7.4), and kepton ice prior to initiating an experiment. The hepatocyte suspensionswere pre-incubated in a shaking water bath at 37° C. for 3 minutes, andthen the reaction was initiated by addition of Compound A into thehepatocyte suspensions (1.5×106 cells/mL) at a final concentration of 3μM, and a DMSO content of 0.1%. The reaction mixture was incubated in ashaking water bath at 37° C. Aliquots of the incubation solutions weresampled at 0, 15, 30, 60, and 120 minutes. The reaction was immediatelyterminated by the addition of three volumes of ice-cold acetonitrilecontaining 0.1% formic acid and internal standards. After centrifugationat 1640×g for 10 minutes, the supernatants were transferred into HPLCvials, and the test compound was analyzed by LC-MS/MS. Positivecontrols, testosterone (20 μM) and 7-hydroxycoumarin (100 μM), wereperformed in parallel to confirm the enzyme activities of thehepatocytes used.

Experimental half-lives were transformed to the corresponding intrinsicclearance values and subsequently scaled to predicted in vivo clearancevalues, using the well-stirred model as previously described (Houston,Biochemical Pharmacology 47(9): 1469-1479, 1994; Obach, Drug Metabolismand Disposition 27(11): 1350-1359, 1999), with appropriatespecies-specific scaling factors (Barter et al., Current Drug Metabolism8(1): 33-45, 2007).

Plasma Protein Binding, Blood Partitioning and Plasma Stability Assays

Plasma protein binding was assessed by equilibrium dialysis, utilizingthe HT-dialysis cell format with a cellulose semi permeable membrane(molecular weight cut off of 5000 Da). Plasma was warmed to 37° C. andadjusted to pH 7.4 before use. Male Sprague-Dawley rat, male Beagle dog,male CD-1 mouse and mixed sex human plasma was used for the studies. A 5μM test compound solution was prepared in isotonic phosphate buffer andrat, dog, mouse and human plasma (final DMSO concentration of 0.5%). Theplasma-containing solution was introduced to one side of the membrane,and the plasma-free on the other. Incubations were performed for 2-16hours in duplicate in order to allow the compound to reach equilibrium.Haloperidol was included as the control compound for each species. Atthe end of the equilibration time the cells were emptied. Followingprotein precipitation, the samples were centrifuged and analyzed byLC-MS/MS. The samples from the protein containing compartment werequantified using calibration standards prepared in plasma and theprotein free compartments were quantified using calibration standardsprepared in dialysis buffer.

For blood partitioning, Male Sprague Dawley rat blood was sourced fromHarlan Sera-Lab Limited, Loughborough, UK. The haematocrit was measuredusing a Hettich Haematokrit 210 and calculated as the percentage ofpacked cell volume compared to total volume of whole blood. Compound A(final test compound concentration 0.5 μM, final DMSO concentration0.05%) was incubated separately with fresh heparinized whole blood,reference red blood cells and reference plasma for 60 min at 37° C.Following incubation, the whole blood cell samples were centrifuged for5 min at 5,000 g at 4° C. The spiked reference plasma was stored on iceduring this period. The spiked reference red blood cells were freezethawed quickly three times to assist in lysing the red blood cells.Following centrifugation of the whole blood experimental sample, analiquot was sampled from the plasma and red blood cell layers foranalysis. As before, the red blood cell layer was freeze thawed quicklythree times to lyse the red blood cells. After protein precipitation andcentrifugation, the supernatants for the experimental samples andreference samples were analyzed by LC-MS/MS. Blood-to-plasma ratios werecalculated as previously described (Hinderling, Pharmacological Reviews49(3): 279-295, 1997).

For plasma stability, Compound A (1 μM) was incubated with pooled lotsof human, Beagle dog Sprague Dawley rat and CD-1 mouse plasma for 0, 15,30, 60 and 120 min at 37° C. Samples were quenched in methanol andanalyzed by LC-MS/MS analysis.

MDCK Cell Permeability Assays

Confluent monolayers of MDCK or MDR1-MDCK (P-glycoprotein) cells, 7-14days old, in Transwell® dual-chamber plates, with apical and basolateralpH 7.4 were dosed on the apical side (A-to-B) or basolateral side(B-to-A) with Compound A (10 uM) and incubated at 37° C. with 5% CO2 ina humidified incubator. Samples were taken from the donor and receiverchambers at 120 minutes. Each determination was performed in duplicate.Co-dosed lucifer yellow flux was also measured for each monolayer toensure cell monolayers remained intact during the incubation. Allsamples were assayed by LC-MS/MS.

Compound Identification

Compound A was incubated with liver microsomes of various species(mouse, rat, dog or human). In vitro metabolite profiling andidentification were conducted after incubating Compound A (finalconcentration of 10 μM) with mouse, rat, dog or human liver microsomes(final protein concentration of 0.5 mg/mL) at 37° C. in 100 mM potassiumphosphate buffer containing 2 mM Mg²⁺ in the presence of NADPH and UDPGA(with addition of 0.1 mg/mL alamethacin to human and rat microsomes).For all liver microsomal incubations, samples were taken at 0 and 20minutes. All samples were quenched by using acetonitrile/methanolsolution and analyzed using an LC-MS/MS Q-Trap system (AB Sciex,Framingham, Mass.).

The major metabolites of Compound A in terms of the mass spectrometryresponse were identified by comparison of the LC-MS total ionchromatograms (TIC) of 0 minute and 20 minute samples in full scan modeusing LightSight™ 2.0 software. The corresponding product ion tandemmass spectra of Compound A and its metabolites were obtained by usingenhanced product ion (EPI) scans during positive ion electrospray. Thepossible chemical structures of the metabolites were deduced based ontheir MS1 and MS2 spectra.

Prediction of Human Pharmacokinetics Using Preclinical Species

PK endpoints of CL and VDss in mouse, rat and dog were used to scale tothe corresponding parameters in human using a variety of allometric andinterspecies scaling approaches as previously reported (Boxenbaum,Journal of Pharmacokinetics and Biopharmaceutics 10(2): 201-227, 1982;Jones et al., Journal of Pharmaceutical Sciences 100(10): 4074-4089,2011; Lombardo et al., J Clin Pharmacol 53(2): 178-191, 2013a; Lombardoet al., J Clin Pharmacol 53(2): 167-177, 2013b; Mahmood & Balian,Xenobiotica 26(9): 887-895, 1996; Ring et al., Journal of PharmaceuticalSciences 100(10): 4090-4110, 2011; Tang & Mayersohn, Journal ofPharmaceutical Sciences 95(8): 1783-1799, 2006; Tang & Mayersohn, DrugMetabolism and Disposition 33(9): 1297-1303, 2005; Vuppugalla et al.,Journal of Pharmaceutical Sciences 100(10): 4111-4126, 2011). The likelypredictive accuracy of each method was assessed based on visualinspection of the data fit, as well as values such as the R² and theallometric exponent, where appropriate.

In Vivo Pharmacokinetics

The pharmacokinetics of Compound A was studied following IV bolusadministration to mouse, rat and dog as well as following POadministration to mouse and rat. The time-concentration data is shown inFIG. 1 and the parameters derived from non-compartmental analysis aredisplayed in Table I below.

TABLE I Pharmacokinetic parameters in preclinical species after IV andPO administration of Compound A CD-1 Mouse SD Rat Beagle Dog ParameterIV bolus PO IV bolus IV infusion PO IV bolus n 3 3 3 6 3 3 Dose (mg/kg)5 20 1 4.7 (/day) 10 1 Cmax (uM) 7.99 ± 1.90 0.0019 2.04 ± 0.23 0.130.0007 5.06 ± 0.60 t max (h) 0.05 0.5 0.05 4 0.25 0.083 AUC_(0-t) 1.95 ±0.28 0.0014 0.43 ± 0.03 12.5 n.d. 1.55 ± 0.16 (uM · h) AUC_(0-inf) 1.96± 0.30 n.d. 0.44 ± 0.03 12.5 n.d. 1.60 ± 0.15 (uM · h) t½ (h) 1.14 ±0.35 n.d. 3.73 ± 1.03 n.d. n.d. 13.6 ± 2.8  MRT (h) 0.35 ± 0.06 n.d.0.41 ± 0.10 n.d. n.d. 2.17 ± 0.89 CL (mL/min/kg) 76.7 ± 11.5 67.8 ± 5.3 18.7 ± 1.7  VDss (L/kg) 1.58 ± 0.23 1.66 ± 0.42 2.44 ± 1.11 F(%) <1 <1

In mouse, rat and dog the plasma clearance was 77, 68 and 19 mL/min/kgrespectively, which equates to an extraction ratio of 86, 97 and 61%respectively (based on total CL being entirely hepatic and usingspecies-specific liver blood flows of 90, 70 and 31 mL/min/kgrespectively). Volume of distribution at steady state was measured at1.58, 1.66 and 2.44 L/kg in mouse, rat and dog respectively. Inphysiological terms, this corresponds to about 2.2-, 2.4- and 3.5-foldgreater than total body water (0.7 L/kg) respectively indicatingpartitioning into peripheral tissue compartments. The kinetics followingIV bolus administration in all three species showed bi-exponentialdecline, as evidenced by a mean residence time that was shorter than theterminal elimination half-life (Table I). Following PO administrationthe exposure in terms of Cmax, AUC and oral bioavailability was low. Inmouse and rat, following PO administration the exposure in terms ofCmax, AUC and oral bioavailability was low.

Based on the PK/PD relationship, the disease indication and as aconsequence of the low oral exposure of Compound A, the compound wasexplored for the potential delivery by IV infusion. Thetime-concentration profile following a 7 day IV infusion of Compound Ain SD rat is shown in FIG. 2 and the derived parameters are presented inTable I. A dose rate of 4.7 mg/kg/day was able to maintain an averagesteady state concentration of 78 nM. The time to steady state wasachieved within 1 h, in line with the generally accepted 3-5 effectivehalf-lives (Boxenbaum & Battle, Journal of Clinical Pharmacology 35(8):763-766, 1995). Utilizing the dose rate of 4.7 mg/kg/day and the steadystate concentration of 78 nM gives rise to a CL estimate of 74mL/min/kg, which compares favorably with the CL of 68 mL/min/kg derivedfrom intravenous bolus administration.

In Vitro Metabolic Stability

A summary of the metabolic stability data across species is shown inTable II below.

TABLE II Liver microsome stability, hepatocyte stability, scaled hepaticCL and blood and plasma binding across species for Compound A SpeciesMouse Rat Dog Human Liver Microsomal CLint 150 72 39 88 (uL/min/mg)Scaled Hepatic CL from 78 45 20 17 liver microsomes (mL/min/kg) ScaledHepatic CL from 43 23 9 8 liver microsomes w/ fu correction (mL/min/kg)Hepatocyte CLint 3.5 1.5 14.9 <1 (uL/min/million cells) Scaled HepaticCL from 26 7 21 <3 hepatocytes (mL/min/kg) Plasma free fraction 0.1380.272 0.234 0.125 Blood:plasma ratio 2.15 0.77 1.16 0.65 Half-life in exvivo blood >120 >120 >120 >120 plasma (min)

Liver microsomal incubations supplemented with NADPH showed moderateturnover in mouse, rat, dog and human which when scaled by thewell-stirred liver model gave hepatic CL values of 78, 45, 20 and 17mL/min/kg indicating moderate to high hepatic extraction in mouse, rat,dog and human respectively. Incorporating plasma protein binding intothe microsomal scaling gave hepatic CL values of 43, 23, 9 and 8mL/min/kg in mouse, rat, dog and human respectively. In liver microsomalpreparations supplemented with UDPGA and alamethacin, no turnover wasobserved indicating glucuronidation is not a primary metabolic pathwayfor Compound A. In hepatocyte suspensions, turnover of Compound A wasvery low giving rise to low CL estimates in all species tested, with theexception of dog where an hepatic CL value of 21 mL/min/kg was observed.

Plasma Protein Binding and Blood:Plasma Partitioning

The in vitro binding and partitioning data is shown in Table II. Thefree fraction in plasma for Compound A did not show any marked speciesdifferences with values of 0.138, 0.272, 0.234, and 0.125 in mouse, rat,dog and human respectively. The blood-to-plasma partitioning data acrossspecies did not suggest any significant binding of Compound A toerythrocytes with values suggesting a fairly equal distribution betweenplasma and blood components. Based on this data, plasma clearance,rather than blood clearance, was used in all further data analysis.

Permeability in MDCK Cell Monolayers

The permeability of Compound A in native and MDR1-transfected MDCK cellmonolayers is shown in Table III below.

TABLE III Permeability of Compound A across MDCK cell monolayers MeanPapp Efflux Relative Cell line Direction (×10⁻⁶ cm/s) ratio Efflux ratioMDCK - native Apical-to- 0.09 3.3 <2 basolateral Basolateral-to- 0.30apical MDCK - MDR1 Apical-to- <0.06 >3.8 transfected basolateralBasolateral-to- 0.22 apical

Compound A shows low apical-to-basolateral permeability in both celllines with mean Papp values of less than 0.1×10-6 cm/s. The relativeefflux ratio between the transfected and native cell lines suggestsCompound A is not a substrate for P-gp. However, both cell linesindicate an efflux ratio of approximately 3, suggesting the action of anative transporter protein in the basolateral-to-apical efflux ofCompound A.

Interspecies PK Scaling

A summary of interspecies PK predictions for human CL and VDss are shownin Table IV below.

TABLE IV Predicted Human PK parameters using various interspeciesscaling approaches Predicted human CL Predicted human Method (mL/min/kg)VD_(SS) (L/kg) Simple allometry 13.3 (exponent 0.76) 2.71 Simpleallometry - unbound 5.7 1.16 RoE - MLP 5.2 FCIM allometry 4.3 MLRrat-dog 10.7 Oie-Tozer equation 0.97 Wajima equation 6.20 Consensusprediction¹ 6.5 2.76 ¹consensus prediction excludes simple allometryprediction for CL given the exponent falls outside considered acceptablelimits of 0.55 < b < 0.7.

Exemplar allometric plots are illustrated in FIG. 3. Mouse, rat and dogdata were utilized unless otherwise stated. Using simple allometry forCL gave rise to an exponent outside the generally accepted criterion of0.55<b<0.7, and in this case invokes the use of the maximum lifepotential (MLP)×CL product term, as part of the Rule of Exponentsapproach (Mahmood & Balian, 1996). Consistent predictions for human CLwere obtained with simple allometry of unbound CL, MLP.CL and the fucorrected intercept method, all approximately 5 mL/min/kg. Thepreviously reported multiple linear regression approach using rat anddog data gave a human CL prediction of 11 mL/min/kg. The consensusprediction of all 4 interspecies CL scaling approaches was 6.5mL/min/kg.

For VDss, simple allometry using total and unbound terms gave rise tosimilar estimates of 1.2-2.7 L/kg. Using rat and dog data and theOie-Tozer equation (Oie & Tozer, J Pharm Sci 68(9): 1203-1205, 1979)gave a predicted human VDss of 0.97 L/kg whilst the Wajima MLR approach(Wajima et al., Journal of Pharmacy and Pharmacology 55(7): 939-949,2003) using the same preclinical data gave a predicted human VDss muchhigher than both allometry and the Oie-Tozer equation, at 6.2 L/kg. Theconsensus prediction of all 4 interspecies VDss scaling approaches was2.8 L/kg.

Time-Invariant PK Scaling

The time-concentration profiles from IV bolus administration to mouse,rat and dog were scaled using time-invariant PK scaling approaches; theDedrick method which normalizes the time-concentration scales by BŴ0.25and dose/BW respectively, and the Wajima method which normalizes thetime-concentration scale by MRT and Css respectively. The normalizationfor physiological time across species has shown utility in predictingthe PK profile in human (Dedrick et al., Cancer Chemotherapy Reports,Part 1 54(2): 95-101, 1970; Wajima et al., Journal of PharmaceuticalSciences 93(7): 1890-1900, 2004). Using the Wajima approach, thepreclinical time-conc profiles overlaid and showed reasonable congruence(FIG. 4) whilst the Dedrick approach did not show alignment acrossspecies. Back-calculation of the Wajima normalized time-course with thehuman predicted MRT and Css provided a time-concentration PK profile forintravenous dosing in man.

Structural Elucidation of the Major Metabolites of Compound a by LC-MSand LC-MS/MS

The metabolism of Compound A was studied in vitro in liver microsomessupplemented with NADPH and UDPGA, with several metabolites detected inmouse, rat, dog and human. LC-MS and LC-MS/MS were used foridentification of Compound A and its metabolites. The molecular ions andcharacteristic fragment ions are exemplified in FIGS. 6-9. A summary ofthe metabolites identified is presented in Table V below and theproposed metabolic pathway is shown in FIG. 10. See, also,Basavapathruni et al., Biopharmaceutics & Drug Disposition, 35: 237-252(2014), the content of which is hereby incorporated by reference in itsentirety.

TABLE V Summary of metabolites of Compound A generated in livermicrosomes supplemented with NADPH and UDPGA in various species. MassRetention Abundance based on shift time UV spectra (%)* Metabolite (Da)m/z (min) Mouse Rat Human Parent 0 563.2 18.5 25.4 59.4 59 M1 +16 579.316.3 4.5 11.3 2.6 M3 +16 579.3 17.2 6.7 3.5 M4 −42 521.3 17.4 M5 +16579.3 17.8 3.8 10.3 M6 +16 579.3 18.0 9.3 *It is not possible toaccurately quantify metabolites in dog by UV due to low signal.

Compound A

The protonated molecular ion of Compound A was m/z 563. The proposedfragmentation pathway is shown in FIG. 6D. Loss of the adenine ring gavem/z 428, with m/z 136 corresponding to the protonated adenine ringitself. Loss of the adenosine moiety gave m/z 326, due to the neutralloss of both the tetrahydrofuran and adenine ring systems. Cleavage atthe N-cyclobutyl bond gave rise to m/z 255 corresponding to theprotonated t-butyl-benzimidazole-cyclobutyl portion of Compound A.Metabolites showed similar fragmentation pathways, which allowed theelucidation and assignment of metabolite structures.

Compound M1

The protonated molecular ion of M1 was m/z 579 indicating a mass shiftof +16 Da and a mono-hydroxylation of Compound A. The proposedfragmentation pathway is shown in FIG. 7A. Comparison of the MS2 data ofM1 with that from the parent compound suggested the mono-hydroxylationoccurred on the t-butyl-benzimidazole-cyclobutyl portion of themolecule, which was further verified by NMR data. This was supported bythe fragment ions from M1 with m/z 444, 342, 330 and 271 all retaining a+16 Da mass shift with corresponding ions from Compound A (m/z 428, 326,314 and 255). The fragment ion m/z 136, corresponding to the adeninering, was present in MS2 spectra for both parent and M1.

Compound M3

The protonated molecular ion of M3 was m/z 579 indicating a mass shiftof +16 Da and a mono-hydroxylation of Compound A. The proposedfragmentation pathway is shown in FIG. 7A. The MS2 data for M3 gavefragment ions of m/z 444, 342 and 271, and as such could not bedifferentiated structurally from M5.

Compound M4

The protonated molecular ion of M4 was m/z 521 indicating a mass shiftof −42 Da and dealkylation of the N-isopropyl group of Compound A. Theproposed fragmentation pathway is shown in FIG. 8A. Comparing the MS2data with that of parent revealed fragment ions m/z 255 and 136 wereidentical in both species, while product ions m/z 386, 368, 350 and 284supported N-dealkylation of the isopropyl group.

Compound M5

The protonated molecular ion of M5 was m/z 579 indicating a mass shiftof +16 Da and a mono-hydroxylation of Compound A. The proposedfragmentation pathway is shown in FIG. 7A. The MS2 data for M5 gavefragment ions of m/z 444, 342 and 271, and as such could not bedifferentiated structurally from M3.

Compound M6

The protonated molecular ion of M6 was m/z 579 indicating a mass shiftof +16 Da and tentatively assigned as an N-oxidation product of CompoundA. The proposed fragmentation pathway is shown in FIG. 9A. The MS2product ions of m/z 428, 326 and 255 were characteristic of thebenzimidazole, cyclobutyl and ribose moieties of the parent moleculesuggesting oxidation of the adenine ring, most likely the N-oxide.

The in vivo PK profiles in mouse, rat and dog following IV bolusadministration showed biexponential kinetics that was more apparent asthe order of the species increased. This resulted in eliminationhalf-lives increasing from 1.1 h in mouse, 3.7 h in rat and 13.6 h indog. In addition, MRT was shorter than the elimination t1/2 furthersupporting multi-exponential kinetics in the preclinical species. The CLin all species was moderate to high with estimated hepatic extractionratios of 86, 97 and 61% in mouse, rat and dog respectively. ExpressingCL in its unbound or blood form did not contribute to any appreciablespecies differences since there was reasonable agreement across speciesin plasma free fraction and with blood partitioning values around unity.Volume of distribution at steady state (VDss) was consistent acrossspecies with values 2-3 fold greater than total body water indicatingpartitioning into peripheral tissues. Unbound VDss was also fairlyconsistent across species at 11.4, 6.1 and 10.4 L/kg in mouse, rat anddog respectively. The intrinsic clearance data in liver microsomes andhepatocytes indicated oxidative metabolism was an important metabolicpathway. The scaled clearance from liver microsomes showed excellentagreement with in vivo clearances in the preclinical species, furthersupporting perfusion-limited CL and oxidative metabolism as the primaryelimination pathway. Interestingly, with the exception of dog, thescaled hepatocyte data gave rise to much lower values between 3- and10-fold lower than observed CL, suggesting permeation or hepatocyteuptake was rate limiting. This has been demonstrated for other compoundsshowing a similar disparity between liver microsome and hepatocyteclearance (Di et al., European Journal of Medicinal Chemistry 57:441-448, 2012). Dog is an apparent outlier in this cross speciescomparison, showing good concordance in clearance prediction by bothliver microsomes and hepatocytes. This may be indicative of an hepaticuptake process specific to the dog, not present in other species wherelow passive permeation limited the turnover in hepatocyte incubations.This is supported by the slightly higher VDss observed in dog whichwould correspond to greater tissue permeation and uptake. Overall, theseobservations are in line with physicochemical properties such as PSA(144 Å²) and the MDCK permeability which was less than 1×10⁻⁶ cm/s.Further in vitro metabolism studies confirmed no evidence ofglucuronidation in all species tested and no instability in bloodplasma. Although there was no indication of Compound A acting as a P-gpsubstrate, there was evidence of a transporter native to the MDCK cellline involved in a basolateral-to-apical efflux process. This is an asyet unidentified transporter and it is noteworthy that it is native tothis cell line of dog kidney origin.

Compound A showed negligible oral bioavailability in mouse and rat,which is in line with the physicochemical properties, in terms ofparameters generally regarded as necessary for favorablegastrointestinal absorption (van de Waterbeemd, Methods and Principlesin Medicinal Chemistry 40 (Drug Bioavailability): 71-99, 2009). CompoundA has a calculated log P of 3.26, a PSA of 144 Å² and a molecular weightof 563. The oral absorption is permeability-limited based on poorpassive permeation observed in MDCK cell monolayers. The oral exposuremay also be perturbed by moderate-to-high first pass extraction inrodents. An IV infusion dosing paradigm was pursued. Pharmacokineticdata in rat demonstrated that steady-state concentrations (Css) could beachieved rapidly, and sufficiently maintained over the dosing interval.Clearance estimates based on the IV infusion data (using dose ratedivided by Css) were in excellent agreement with those from IV bolusadministration; 74 and 68 mL/min/kg respectively.

The predictive accuracy of interspecies PK scaling has received muchattention recently with several large initiatives to identify the mostpredictive methods in estimating human PK endpoints from preclinicalspecies (Jones et al., 2011; Lombardo et al., 2013a; Lombardo et al.,2013b; Poulin et al., Journal of Pharmaceutical Sciences 100(10):4050-4073, 2011a; Poulin et al., Journal of Pharmaceutical Sciences100(10): 4127-4157, 2011b; Ring et al., 2011; Vuppugalla et al., 2011).As shown in Table IV, the data were analyzed in several ways using amultitude of approaches recommended in these recent analyses, givingcareful consideration to cases of congruence or disparity in prediction.Three orthogonal methods were utilized including IVIVE, interspeciesscaling and time-invariant scaling. Simple allometry utilizing total andunbound VDss gave rise to similar predictions as did the use of theOie-Tozer equation, all indicating a human VDss in the 1-3 L/kg range.The recent PhRMA analysis and others have shown the superior predictiveaccuracy afforded by the Oie-Tozer approach (Oie & Tozer, 1979). ForCompound A, allometric and physiologically-based methods gave similarpredictions for VDss. The Wajima equation which utilizes a multiplelinear regression of rat and dog VDss gave a much higher estimate ofhuman VDss (Wajima et al., 2003). This may be a result of the MLRequation being weighted towards dog VDss, which for Compound A was thehighest VDss observed in preclinical species. Simple allometry of CLgave rise to an exponent outside the acceptable range of 0.55<b<0.7, andas a result the rule of exponents was adopted and the product term of CLand maximum life potential was utilized (Mahmood & Balian, 1996). Theinterspecies scaling of CL was attained by the approaches of simpleallometry using unbound CL, Rule of Exponents using MLP, fu-correctedintercept method allometry and a multiple linear regression method usingrat and dog CL, all giving rise to a consensus prediction of 6.5mL/min/kg for human CL and with a reasonably low CV (44%) across thedifferent methods. Recent global analyses of human CL prediction bydifferent groups have highlighted the predictive accuracy of thefu-corrected intercept method (Lombardo et al., 2013a; Ring et al.,2011). The allometric methods show very good agreement and are expectedto perform well in cases such as this, where compounds exhibit moderateCL across species.

Liver microsomes supplemented with NADPH provided good agreement with invivo CL across all three preclinical species. Even in the case ofincorporating plasma protein binding into the well stirred model, the CLestimates remained within 2-3 fold of the measured CL. Challenges andlimitations in IVIVE include whether to incorporate plasma free fractionwhen the level of protein binding is low to moderate and may introduce afold change in CL coincident with the current practical limit inpredictive accuracy for IVIVE of 2-3 fold. That said, liver microsomalCL with fu correction does provide a human CL estimate in good agreementwith the interspecies scaling methods. In addition, an observation madeby the recent PhRMA initiative eluded to the greater predictive accuracyof in vivo scaling methods compared to current IVIVE approaches. Thismay relate to specific dynamic and complex equilibria present in vivothat are not captured in the simplified in vitro setting. In addition,it is a common observation that liver microsomes have a tendency tooverpredict CL especially for compounds with low passive membranepermeability. This was largely exemplified with the scaled hepatocyte CLvalues for Compound A in which low turnover was observed for mouse, ratand human (CLint<4 uL/min/million cells). Dog was a clear outlier interms of scaled hepatocyte clearance and this may relate to a hepaticuptake process well represented in dog. Overall, across multiple,diverse scaling approaches Compound A human CL estimates are in therange of 6-8 mL/min/kg. The time-invariant PK methodology originallyreported by Wajima showed a compelling overlay of the preclinical datawhen normalized for time (t/MRT) and concentration (C/Css) and provideda useful transformation of the primary human PK parameters into atime-concentration plot with which to model various clinical dosingparadigms.

Due to the much lower turnover observed in hepatocytes, liver microsomeswere selected for metabolite identification and profiling. As notedearlier, no turnover was observed in blood plasma or liver microsomessupplemented with alamethacin and UDPGA, indicating that hydrolysis orglucuronidation were not primarily involved in the metabolism ofCompound A. In liver microsomes supplemented with NADPH and UDPGA,several oxidative metabolites were observed. Metabolites M1, M3 and M5were all distinct mono-hydroxylations on thebenzimidazoles-ethylene-cyclobutyl portion of the molecule. Thehydroxylation position of M1 (as shown in FIG. 10) was verified by NMRexperiment. The exact positions of hydroxylations in M3 and M5 are yetto be determined. M3 was only observed in rat whilst M1 and M5 werepresent in all preclinical species as well as human. Metabolite M4,N-dealkylation and loss of the isopropyl group, was observed in allspecies tested whilst M6, the N-oxidation of the adenine ring, was onlyobserved in rat. No glucuronides of Compound A or its hydroxylatedmetabolites were detected and no metabolites unique to human werepresent in this in vitro metabolism study.

The biological activities of the compounds of the invention can beevaluated using the methods described in Examples 3-8 below.

Example 3: General Methods

Cell Culture:

Human leukemia cell line EOL-1 (Catalog # ACC-386) is purchased fromDSMZ and are grown in Roswell Park Memorial Institute medium (RPMI) with10% Fetal Bovine Serum (FBS). Cells are kept in log growth as outlinedin the technical data sheet provided by the vendor.

Human hematological tumor cell lines THP-1, RS4; 11, and MV4-11 areobtained from ATCC, MOLM-13 cells are obtained from DSMZ. All lines aregrown in RPMI 1640 containing 10% FBS and maintained using the vendorsrecommended cell densities and environmental conditions. Media issupplemented with non-essential amino acids and L-Glutamine. THP-1 cellsare also supplemented with 0.05 mM 13-Mercaptoethanol.

Methylation Analysis.

Cells are seeded at 5×10⁵ cells/mL in a 12 well plate at a final volumeof 2 mLs. Cells are dosed with compounds to the appropriateconcentration from a 50 mM DMSO stock solution. Compound and media arerefreshed every two days over the course of seven day incubation bycounting cells using trypan blue exclusion (Vicell), pelleting at 200 gfor 5 minutes and resuspending in fresh media containing compound at afinal cell concentration of 5×10⁵ cells/mL. Following compoundincubation, histones are extracted from 1×10⁶ cells using a commercialhistone extraction kit (Active Motif). Purified histones are quantitatedusing the BCA protein assay (Pierce) with a BSA standard curve. 400 ngof isolated histones are fractionated by SDS-PAGE on a 4-20% gel andtransferred to nitrocellulose membranes. Membranes are incubated withvarious primary and secondary antibodies and imaged on the Licor imagingsystem (Odyssey). The H3K79-Me2 rabbit polyclonal is purchased fromAbcam. Other rabbit polyclonal antibodies including H3K4-Me3, H3K9-Me3,H3K27-Me2, and H3K27-Me3 are purchased from Cell Signaling Technologies(CST). A mouse monoclonal total H3 antibody is used as a loading control(CST). Fluorescently labeled secondary antibodies are purchased fromOdyssey.

Cell Growth and Viability Assay

Exponentially growing cells (e.g., EOL-1, THP-1, MV4-11 and MOLM-13cells) are plated in 96-well plates at a density of 3×10⁴ viablecells/well. Each treatment is seeded in triplicate with a final wellvolume of 150 μLs. Cells are incubated with increasing concentrations ofDOT1L inhibitor up to 50 μM. Viable cell number is determined every 3-4days for 11 days using the Guava Viacount assay (Millipore #4000-0040)and analyzed on a Guava EasyCyte Plus instrument according to themanufacturer's protocol. On the days of cell counts, growth media andinhibitor are replenished and cells maintained in log phase culture byreseeding at a density of 5×10⁴ viable cells/well. Total cell number isexpressed as split-adjusted viable cells per well. For each cellinhibitor IC₅₀ values are determined from concentration-dependencecurves at day 11. All calculations are done using GraphPad Prism,version 5.00 for Windows, GraphPad Software, San Diego Calif. USA(www.graphpad.com).

Histone Extraction of Cell Pellets:

Frozen pellets are allowed to thaw briefly on ice and then lysed by a 5minute incubation on ice with 250 μl nuclear extraction buffer (10 mMTris-HCl, pH 7.6, 10 mM MgCl₂, 25 mM KCl, 1% Triton X-100, 8.6% Sucrose,plus a Roche protease inhibitor tablet 1836153001). Nuclei are collectedby centrifugation at 600 g for 5 minutes at 4° C. and washed once inTris/EDTA buffer (pH 7.4). Supernatant is removed and histones extractedfor one hour with 60 μl 0.4 N cold sulfuric acid. Extracts are clarifiedby centrifugation at 10,000 g for 10 minutes at 4° C. and transferred toa fresh microcentrifuge tube containing 600 μl ice cold acetone.Histones are precipitated at −20° C. for 2 hours, pelleted bycentrifugation at 10,000 g for 10 minutes and resuspended in 60 μldistilled water (DI water). Total protein of the acid extracts isassessed using a bicinchoninic acid (BCA) protein quantification assaywith a bovine serum albumin (BSA) standard (Pierce Biotechnology).

H3K79me2 Immunoblot:

For immunoblot analysis of the H3K79me2 inhibition by DOT1L inhibitor,exponentially growing cells (e.g., EOL-1 cells) are seeded at 2×10⁵cells/mL and incubated in the presence of increasing concentrations ofDOT1L inhibitor for 4 days. Following incubation, cells (2-3×10⁶) areharvested and histones extracted as described. Histones (400 ng) arefractionated on a 10-20% Tris HCl gels (Bio-Rad) with Tris-Glycine SDSrunning buffer (Invitrogen) under denaturing conditions and transferredto a nitrocellulose filter. The filter is incubated for 1 hour inblocking buffer (Odyssey blocking buffer, Li-cor, 927-40000) at RT andthen incubated overnight at 4° C. in blocking buffer containing anantibody specific for H3K79me2 (1:5000 dilution, abcam ab3594). Filtersare washed 3 times for 5 minutes with wash buffer (PBST) and incubatedwith infrared tagged secondary antibody (Alexa Flour 680 goatanti-rabbit IgG (1:20,000), Invitrogen A-21076) at RT for 1 hour.Filters are washed in PBST and reprobed for 1 hour at RT with theappropriate total histone antibody control (mouse anti-histone H3(1:20,000), CST 3638, or mouse anti-histone H4 (1:10,000), CST 2935).Filters are washed again in PBST and incubated with infrared taggedsecondary antibody (IRDye 800Cw donkey-anti-mouse IgG (1:20,000), Li-Cor926-32212) at RT for 1 hour. After a final wash in PBST, filters arescanned using the Odyssey infrared imager (Li-cor). Signal intensitiesspecific for each methyl-specific antibody is quantified using Odysseysoftware and normalized to that of the appropriate total histone controlsignal on the same filter by dividing the methyl-specific antibodysignal intensity by the total histone control signal intensity.

Quantitative Real-Time PCR:

Exponentially growing cells (e.g., EOL-1 cells) are plated in a 12 wellplate at 2×10⁵ cells/mL. Cells are incubated in the presence ofincreasing concentrations of a test compound up to 10 μM. On day 4,cells are maintained in log phase culture by reseeding at 5×10⁵ cells/mLand compound is replenished. At day 6 cells are washed twice with PBSand pelleted by centrifugation at 200×g. Cell pellets are lysed in 300μL RLT buffer (Qiagen) and total RNA is isolated using the RNeasy totalRNA isolation kit (Qiagen 74106). Total RNA (1 μg) was reversetranscribed using a high capacity cDNA reverse transcription kit(Applied Biosystems 4368813). RNA isolation and cDNA synthesis arecarried out according to the manufacturer's protocol. Predesignedlabeled primer and probe sets for HOXA9 (Hs00365956), MEIS1 (Hs00180020)and FLT3 (Hs00975659) are purchased from Applied Biosystems.Quantitative real-time PCR (qPCR) reactions contained 50 ng cDNA, 1×labeled primer and probe set, and 1× Taqman universal PCR master mix(Applied Biosystems 4304437). Samples are run on a 7900 HT Fast RealTime PCR machine (Applied Biosystems 4351405) with cycling conditions of2 min 50° C., 10 min 95° C., 40 cycles at 15 sec 95° C. and 1 min 60° C.Target gene cycle numbers are normalized to the house keeping gene2-microglobulin (Applied Biosystems 4333766) to get a ACT value. Percentof DMSO control is calculated with the equation (2^(−ΔΔCT))*100 wherethe ΔΔCT is the difference between normalized target gene and DMSOcontrol (ΔCT sample−ΔCT control=ΔΔCT).

Determination of IC₅₀.

Test compounds are serially diluted 3 fold in DMSO for 10 points and 1μl is plated in a 384 well microtiter plate. Positive control (100%inhibition standard) is 2.5 uM final concentration ofS-adenosyl-L-homocysteine and negative control (0% inhibition standard)contained 1 μl of DMSO. Compound is then incubated for 30 minutes with40 μl per well of DOT1L(1-416) (0.25 nM final concentration in assaybuffer: 20 mM TRIS, pH 8.0, 10 mM NaCl, 0.002% Tween20, 0.005% BovineSkin Gelatin, 100 mM KCl, and 0.5 mM DTT). 10 μl per well of substratemix (same assay buffer with 200 nM S-[methyl-³H]-adenosyl-L methionine,600 nM of unlabeled S-[methyl-³H]-adenosyl-L methionine, and 20 nMoligonucleosome) is added to initiate the reaction. Reaction isincubated for 120 minutes at room temperature and quenched with 10 μlper well of 100 μM S-methyl-adenosyl-L methionine. For detection,substrate from 50 μl of reaction is immobilized on a 384 wellStreptavidin coated Flashplate (Perkin Elmer) (also coated with 0.2%polyethyleneimine) and read on a Top Count scintillation counter (PerkinElmer).

Other related general procedures and specific preparation procedures canalso be found in the PCT publication Nos. WO2012/075381 and WO2014/026198, the contents of each are incorporated herein by referencein their entireties.

Example 4: Effect of DOT1L Inhibition on Cell Growth and Viability

The effect of DOT1L inhibitors on leukemia cell growth and viability isinvestigated. EOL-1 cells, a leukemia cell line characterized by MLLPTD, are plated in 96-well plates at a density of 3×10⁴ viablecells/well. Cells are incubated with increasing concentrations of DOT1Linhibitor between the 0.003 μM-50 μM. The number of viable cells isdetermined every 3-4 days for 11 days. Cells are maintained in log phaseby reseeding and replenishing growth media and the indicatedconcentration of DOT1L inhibitor on each day of cell counts (Day 0, Day4, Day 7, and Day 11). Total cell number is expressed as split-adjustedviable cells per well. DMSO-treated cells are used as a control.

Cell proliferation is inhibited in a dosage-dependent manner, with thehighest concentration (50 μM) having the most pronounced inhibitoryeffect on cell proliferation and viability.

Example 5: Inhibition of DOT1L Methyltransferase Activity

Inhibition of methylation of H3K79 is assessed after 4 days of treatmentof DOT1L compounds in exponentially growing cells (e.g., EOL-1 cells).

H3K79 methylation status after treatment with a test compound is firstexamined by immunoblot. Following treatment, cells are harvested andhistones are extracted. Western blot analysis is performed usingantibodies specific for H3K79me2 and total histone 3 (as a control).Signal intensities specific to H3K79me2 is quantified and normalized tothat of the total histone 3 signal.

Example 6: Potency of DOT1L Inhibition

The IC50 and inhibition constants of the DOT1L inhibitors describedherein can be determined from concentration-dependence growth curves, asdescribed in Example 2. The concentration-dependence curves at day 11for a test compound and a control compound are plotted on a single loggraph and compared. IC50 values, or maximal inhibitory concentration,for each DOT1L inhibitor can be determined from the curves.

The inhibition constant (Ki) can also be determined for the DOT1Linhibitors described herein. For example, Ki values for a test compoundand a control compound can be compared. The test compound having thelowest Ki, which indicates that a very low concentration (e.g., 0.08 nM)of the test compound is required to decrease the maximal rate of thereaction to half of the uninhibited value, in the presence of a lowsubstrate concentration. As expected, the control compound has a veryhigh Ki (e.g., more than 50,000 nM), showing that the control compoundrequires a very little inhibitory activity.

Example 7: Gene Overexpression in Leukemias

HOXA9 expression levels are assessed in a panel of leukemia cells lines,demonstrating that HOXA9 is often overexpressed in various hematologiccancers. The leukemia cells assessed include MolM13 (acute monocyticleukemia cell line), MV411 (acute myelocytic leukemia), LOUCY (T-cellacute lymphoblastic leukemia), EOL-1 (eosinophilic leukemia) SemK2(B-cell acute lymphoblastic leukemia), Reh (acute lymphoblasticleukemia), HL60 (promyelocytic leukemia) and BV173 (pre-B-cellleukemia). Molm13, MV411, and SemK2 cell lines are characterized ashaving MLL fusions. LOUCY and Reh cell lines are characterized as havingnon-MLL chromosomal rearrangements. EOL-1 cells are characterized ashaving MLL-PTD.

Cells are harvested, RNA is extracted, and cDNA is prepared as describedin Example 1. The expression level of HOXA9 is determined byquantitative real-time PCR. HOXA9 expression level is normalized to thelowest HOXA9 expresser. Jurkat cells, an immortalized T-lymphocyte cellline, are used as control. HL60, BV173, and Reh cell lines had very lowoverexpression of HOXA9. SemK2, EOL-1, LOUCY, MV411, and Molm13 hadextremely high overexpression of HOXA9, with at least a 2000-foldincrease over the lowest expresser. Overexpression of othercancer-associated genes, such as FLT3, MEIS1, or DOT1L can be determinedusing similar methods as those described herein.

Example 8: Gene Expression after DOT1L Inhibition

Leukemia cell lines can be treated with DOT1L inhibitors and theexpression of select genes can be examined to assess the effects ofDOT1L inhibition on cancer-associated gene overexpression. Leukemia celllines are treated with increasing concentrations of candidate DOT1Linhibitors up to 10 μM for 6 days. For the vehicle control, cells aretreated with DMSO. Cells are then harvested, RNA is extracted, and cDNAis prepared as described in Example 1.

Expression levels of select genes, including HOXA9, FLT3, MEIS1, andDOT1L, are determined by quantitative real-time PCR. Expression ofHOXA9, FLT3 and/or MEIS1 can be reduced in a dose-dependent manner aftertreatment with a test compound. DOT1L gene expression is not reducedafter treatment with DOT1L inhibitor and can be considered useful forcontrol purposes.

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-35. (canceled)
 36. A compound of Formula (II):

or a pharmaceutically acceptable salt or ester thereof, wherein R₁ isunsubstituted t-butyl or t-butyl substituted with one or moresubstituents selected from hydroxyl and oxo; R₂ is H, hydroxyl,unsubstituted i-propyl, or i-propyl substituted with one or morehydroxyl; l′ is 0, 1, 2, or 3; each of m′ and n′, independently, is 0,1, or 2; each of p′, q′, r′, t′, u′, v′, and w′, independently, is 0 or1; and when R₁ is t-butyl substituted with only one hydroxyl, R₂ is H,hydroxyl, or i-propyl substituted with one or more hydroxyl; and when R₁is unsubstituted t-butyl, then (i) R₂ is hydroxyl, or i-propylsubstituted with one or more hydroxyl, or (ii) the sum of l′, m′, n′,p′, q′, r′, t′, u′, v′, and w′ is 1 or greater.
 37. The compound ofclaim 36 or the pharmaceutically acceptable salt or ester thereof, beingin an isolated form.
 38. The compound of claim 36, being of Formula(IIA):

or Formula (IIB):


39. The compound of claim 36, comprising one hydroxyl in addition to thetwo hydroxyls on the tetrahydrofuran ring.
 40. The compound of claim 36,wherein R₁ is unsubstituted t-butyl, and (i) R₂ is hydroxyl or i-propylsubstituted with one hydroxyl, and each of l′, m′, n′, u′, v′, and w′ is0, or (ii) R₂ is H or unsubstituted i-propyl, and the sum of l′, m′, n′,u′, v′, and w′ is
 1. 41. The compound of claim 36, wherein R₁ is t-butylsubstituted with one hydroxyl, R₂ is H, and each of l′, m′, n′, u′, v′,and w′ is
 0. 42. The compound of claim 36, being of a carboxylic acid.43. The compound of claim 36, wherein R₁ is t-butyl substituted with onehydroxyl and one oxo, and wherein the hydroxyl and oxo together with thecarbon to which they are attached form —COOH.
 44. The compound of claim36, wherein R₁ is —C(CH₃)₂COOH), R₂ is H or unsubstituted i-propyl, andeach of l′, m′, n′, u′, v′, and w′ is
 0. 45. The compound of claim 36,comprising two or three hydroxyls in addition to the two hydroxyls onthe tetrahydrofuran ring.
 46. The compound of claim 36, wherein R₁ ist-butyl substituted with one hydroxyl, and (i) R₂ is hydroxyl ori-propyl substituted with one or two hydroxyl, and each of l′, m′, n′,u′, v′, and w′ is 0, or (ii) R₂ is H, hydroxyl, or i-propyl optionallysubstituted with one hydroxyl, and the sum of l′, m′, n′, u′, v′, and w′is
 1. 47. The compound of claim 36, wherein R₁ is t-butyl substitutedwith two hydroxyl, and (i) R₂ is H, hydroxyl or i-propyl optionallysubstituted with one hydroxyl, and each of l′, m′, n′, u′, v′, and w′ is0, or (ii) R₂ is H, hydroxyl or i-propyl optionally substituted with onehydroxyl, and the sum of l′, m′, n′, u′, v′, and w′ is
 1. 48. Thecompound of claim 36, wherein R₁ is t-butyl substituted with threehydroxyl, R₂ is H or unsubstituted i-propyl, and each of l′, m′, n′, u′,v′, and w′ is
 0. 49. The compound of claim 36, comprising four hydroxylsin addition to the two hydroxyls on the tetrahydrofuran ring.
 50. Thecompound of claim 36, comprising five or more hydroxyls in addition tothe two hydroxyls on the tetrahydrofuran ring.
 51. The compound of claim36, wherein the compound is selected from the group consisting ofCompounds 1-89 and 101-104, and 107-114.
 52. A pharmaceuticalcomposition comprising a compound of Formula (II):

or a pharmaceutically acceptable salt or ester thereof, and apharmaceutically acceptable carrier, wherein R₁ is unsubstituted t-butylor t-butyl substituted with one or more substituents selected fromhydroxyl and oxo; R₂ is H, hydroxyl, unsubstituted i-propyl, or i-propylsubstituted with one or more hydroxyl; l′ is 0, 1, 2, or 3; each of m′and n′, independently, is 0, 1, or 2; each of p′, q′, r′, t′, u′, v′,and w′, independently, is 0 or 1; and when R₁ is t-butyl substitutedwith only one hydroxyl, R₂ is H, hydroxyl, or i-propyl substituted withone or more hydroxyl; and when R₁ is unsubstituted t-butyl, then (i) R₂is hydroxyl, or i-propyl substituted with one or more hydroxyl, or (ii)the sum of l′, m′, n′, p′, q′, r′, t′, u′, v′, and w′ is 1 or greater.53. The pharmaceutical composition of claim 52, wherein the compound ofFormula (II) or the pharmaceutically acceptable salt or ester thereof isin an isolated form.
 54. A method of treating cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the pharmaceutical composition of claim
 52. 55. A method oftreating hematological cancer comprising administering to a subject inneed thereof a therapeutically effective amount of the pharmaceuticalcomposition of claim
 52. 56. A method of treating a disorder mediated bya translocation of a gene on chromosome 11q23, comprising administeringto a subject in need thereof a therapeutically effective amount of thepharmaceutical composition of claim
 52. 57. A method of treating adisorder mediated by DOT1L-mediated protein methylation, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of pharmaceutical composition of claim
 52. 58. A method oftreating leukemia comprising administering to a subject in need thereofa therapeutically effective amount of the pharmaceutical composition ofclaim
 52. 59. The method of claim 58, wherein the leukemia is acutemyeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.60. The method of claim 58, wherein the leukemia is characterized by achromosomal rearrangement.
 61. The method of claim 60, wherein saidchromosomal rearrangement is chimeric fusion of mixed lineage leukemiagene (MLL) or partial tandem duplication of MLL (MLL-PTD).
 62. Themethod of claim 61, wherein the subject has an increased level of HOXA9,Fms-like tyrosine kinase 3 (FLT3), MEIS1, and/or DOT1L.
 63. A method fortreating leukemia in a subject comprising: 1) obtaining a sample fromthe subject; 2) (a) detecting the level of HOXA9, FLT3, MEIS1, and/orDOT1L, wherein an increased level of HOXA9, FLT3, MEIS1, and/or DOTILindicates the subject is responsive to the compound of claim 35, or (b)detecting the presence of a genetic lesion of MLL in the sample; and 3)administering to the subject a therapeutically effective amount of thecompound of claim 35 when the subject is responsive to the compound orwhen the genetic lesion is present in the sample.
 64. The method ofclaim 63, wherein the sample is selected from bone marrow, peripheralblood cells, blood, plasma, serum, urine, saliva, a cell, or a tumortissue.
 65. The method of claim 64, wherein the genetic lesion ischimeric fusion of MLL or MLL-PTD.